Isolated human enzyme proteins, nucleic acid molecules encoding human enzyme proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the enzyme peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the enzyme peptides, and methods of identifying modulators of the enzyme peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of enzyme proteins that arerelated to the trans-prenyltransferase subfamily, recombinant DNAmolecules, and protein production. The present invention specificallyprovides novel peptides and proteins and nucleic acid molecules encodingsuch peptide and protein molecules, all of which are useful in thedevelopment of human therapeutics and diagnostic compositions andmethods.

BACKGROUND OF THE INVENTION

[0002] Many human enzymes serve as targets for the action ofpharmaceutically active compounds. Several classes of human enzymes thatserve as such targets include helicase, steroid esterase and sulfatase,convertase, synthase, dehydrogenase, monoxygenase, transferase, kinase,glutanase, decarboxylase, isomerase and reductase. It is thereforeimportant in developing new pharmaceutical compounds to identify targetenzyme proteins that can be put into high-throughput screening formats.The present invention advances the state of the art by providing novelhuman drug target enzymes related to the trans-prenyltransferasesubfamily.

[0003] The present invention has substantial similarity totrans-prenyltransferase (polyprenyl pyrophosphate synthetase).Trans-prenyltransferase synthesizes the side-chain moiety of coenzyme Q.taking place in microsomes of Golgi system. Trans-prenyltransferase, aubiquitous protein, is a member of the coenzyme Q biosynthesis pathway,and a very important enzyme involved in biosynthesis of polyprenyls andcoenzyme Q. CoQ synthesis is also dependent on trans-prenyltransferaseactivity on the level of intracellular substrate concentration. Inaddition, CoQ level may be regulated in blood as well as in varioustissues. Thus, the enzyme of the present invention may be a potentialdrug target for anti-cancer treatment. For a review related totrans-prenyltransferase, see Appelkvist et al., Mol Aspects Med 1994;15Suppl:s37-46; Rotig et al., Lancet Jul. 29, 2000;356(9227):391-5.

[0004] Enzyme proteins, particularly members of thetrans-prenyltransferase subfamily, are a major target for drug actionand development. Accordingly, it is valuable to the field ofpharmaceutical development to identify and characterize previouslyunknown members of this subfamily of enzyme proteins. The presentinvention advances the state of the art by providing previouslyunidentified human enzyme proteins, and the polynucleotides encodingthem, that have homology to members of the trans-prenyltransferasesubfamily. These novel compositions are useful in the diagnosis,prevention and treatment of biological processes associated with humandiseases.

SUMMARY OF THE INVENTION

[0005] The present invention is based in part on the identification ofamino acid sequences of human enzyme peptides and proteins that arerelated to the trans-prenyltransferase subfamily, as well as allelicvariants and other mammalian orthologs thereof. These unique peptidesequences, and nucleic acid sequences that encode these peptides, can beused as models for the development of human therapeutic targets, aid inthe identification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate enzyme activity incells and tissues that express the enzyme. Experimental data as providedin FIG. 1 indicates expression in humans in the T cells from T cellleukemia, B. cells from Burkitt lymphoma, neuroblastoma cells, duodenaladenocarcinoma cell line, adenocarcinoma cell line, stomach, breast,whole liver.

DESCRIPTION OF THE FIGURE SHEETS

[0006]FIG. 1 provides the nucleotide sequence of a cDNA moleculesequence that encodes the enzyme protein of the present invention. (SEQID NO: 1) In addition, structure and functional information is provided,such as ATG start, stop and tissue distribution, where available, thatallows one to readily determine specific uses of inventions based onthis molecular sequence. Experimental data as provided in FIG. 1indicates expression in humans in the T. cells from T cell leukemia, Bcells from Burkitt lymphoma, neuroblastoma cells, duodenaladenocarcinoma cell line, adenocarcinoma cell line, stomach, breast,whole liver.

[0007]FIG. 2 provides the predicted amino acid sequence of the enzyme ofthe present invention. (SEQ ID NO:2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0008]FIG. 3 provides genomic sequences that span the gene encoding theenzyme protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. 57 SNPs, including 8 indels, have been identified in the geneencoding the enzyme protein provided by the present invention and aregiven in FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION

[0009] General Description

[0010] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a enzyme protein or part of aenzyme protein and are related to the trans-prenyltransferase subfamily.Utilizing these sequences, additional genomic sequences were assembledand transcript and/or cDNA sequences were isolated and characterized.Based on this analysis, the present invention provides amino acidsequences of human enzyme peptides and proteins that are related to thetrans-prenyltransferase subfamily, nucleic acid sequences in the form oftranscript sequences, cDNA sequences and/or genomic sequences thatencode these enzyme peptides and proteins, nucleic acid variation(allelic information), tissue distribution of expression, andinformation about the closest art known protein/peptide/domain that hasstructural or sequence homology to the enzyme of the present invention.

[0011] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known enzyme proteins of thetrans-prenyltransferase subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in humansin the T. cells from T cell leukemia, B. cells from Burkitt lymphoma,neuroblastoma cells, duodenal adenocarcinoma cell line, adenocarcinomacell line, stomach, breast, whole liver. The art has clearly establishedthe commercial importance of members of this family of proteins andproteins that have expression patterns similar to that of the presentgene. Some of the more specific features of the peptides of the presentinvention, and the uses thereof, are described herein, particularly inthe Background of the Invention and in the annotation provided in theFigures, and/or are known within the art for each of the knowntrans-prenyltransferase family or subfamily of enzyme proteins.

[0012] Specific Embodiments

[0013] Peptide Molecules

[0014] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of theenzyme family of proteins and are related to the trans-prenyltransferasesubfamily (protein sequences are provided in FIG. 2, transcript/cDNAsequences are provided in FIG. 1 and genomic sequences are provided inFIG. 3). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, particularly allelic variants asidentified herein and using the information in FIG. 3, will be referredherein as the enzyme peptides of the present invention, enzyme peptides,or peptides/proteins of the present invention.

[0015] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the enzyme peptides disclosed in the FIG. 2, (encodedby the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

[0016] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0017] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0018] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of theenzyme peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0019] The isolated enzyme peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in the T cells from T. cell leukemia, B. cells from Burkittlymphoma, neuroblastoma cells, duodenal adenocarcinoma cell line,adenocarcinoma cell line, stomach, breast, whole liver. For example, anucleic acid molecule encoding the enzyme peptide is cloned into anexpression vector, the expression vector introduced into a host cell andthe protein expressed in the host cell. The protein can then be isolatedfrom the cells by an appropriate purification scheme using standardprotein purification techniques. Many of these techniques are describedin detail below.

[0020] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). The amino acid sequence of such a protein is providedin FIG. 2. A protein consists of an amino acid sequence when the aminoacid sequence is the final amino acid sequence of the protein.

[0021] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ IDNO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0022] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the enzyme peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0023] The enzyme peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a enzyme peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the enzyme peptide. “Operatively linked”indicates that the enzyme peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the enzyme peptide.

[0024] In some uses, the fusion protein does not affect the activity ofthe enzyme peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant enzyme peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

[0025] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A enzyme peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the enzyme peptide.

[0026] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0027] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the enzyme peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0028] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0029] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of. 4.

[0030] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0031] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the enzyme peptides of the present invention as well as beingencoded by the same genetic locus as the enzyme peptide provided herein.As indicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 10 by ePCR.

[0032] Allelic variants of a enzyme peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the enzyme peptide as well asbeing encoded by the same genetic locus as the enzyme peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. As indicated by the data presented in FIG. 3, themap position was determined to be on chromosome 10 by ePCR. As usedherein, two proteins (or a region of the proteins) have significanthomology when the amino acid sequences are typically at least about70-80%, 80-90%, and more typically at least about 90-95% or morehomologous. A significantly homologous amino acid sequence, according tothe present invention, will be encoded by a nucleic acid sequence thatwill hybridize to a enzyme peptide encoding nucleic acid molecule understringent conditions as more filly described below.

[0033]FIG. 3 provides information on SNPs that have been found in thegene encoding the enzyme protein of the present invention. SNPs wereidentified at 57 different nucleotide positions in introns and regions5′ and 3′ of the ORF. Such SNPs in introns and outside the ORF mayaffect control/regulatory elements.

[0034] Paralogs of a enzyme peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the enzyme peptide, as being encoded by a gene from humans,and as having similar activity or function. Two proteins will typicallybe considered paralogs when the amino acid sequences are typically atleast about 60% or greater, and more typically at least about 70% orgreater homology through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a enzymepeptide encoding nucleic acid molecule under moderate to stringentconditions as more fully described below.

[0035] Orthologs of a enzyme peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the enzyme peptide as well as being encoded by a gene fromanother organism. Preferred orthologs will be isolated from mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs will be encoded by a nucleic acid sequencethat will hybridize to a enzyme peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0036] Non-naturally occurring variants of the enzyme peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the enzyme peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a enzyme peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

[0037] Variant enzyme peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0038] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0039] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as enzyme activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0040] The present invention further provides fragments of the enzymepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0041] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a enzyme peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the enzyme peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe enzyme peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0042] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inenzyme peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0043] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0044] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

[0045] Accordingly, the enzyme peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature enzyme peptide is fused withanother compound, such as a compound to increase the half-life of theenzyme peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature enzyme peptide, such as aleader or secretory sequence or a sequence for purification of themature enzyme peptide or a pro-protein sequence.

[0046] Protein/Peptide Uses

[0047] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a enzyme-effectorprotein interaction or enzyme-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

[0048] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0049] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, enzymes isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the enzyme. Experimental data as providedin FIG. 1 indicates that the enzymes of the present invention areexpressed in humans in the T cells from T cell leukemia, B cells fromBurkitt lymphoma, neuroblastoma cells, duodenal adenocarcinoma cellline, adenocarcinoma cell line, stomach and breast detected by a virtualnorthern blot. In addition, PCR-based tissue screening panels indicateexpression in whole liver. A large percentage of pharmaceutical agentsare being developed that modulate the activity of enzyme proteins,particularly members of the trans-prenyltransferase subfamily (seeBackground of the Invention). The structural and functional informationprovided in the Background and Figures provide specific and substantialuses for the molecules of the present invention, particularly incombination with the expression information provided in FIG. 1.Experimental data as provided in FIG. 1 indicates expression in humansin the T cells from T cell leukemia, B cells from Burkitt lymphoma,neuroblastoma cells, duodenal adenocarcinoma cell line, adenocarcinomacell line, stomach, breast, whole liver. Such uses can readily bedetermined using the information provided herein, that which is known inthe art, and routine experimentation.

[0050] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to enzymes that are related tomembers of the trans-prenyltransferase subfamily. Such assays involveany of the known enzyme functions or activities or properties useful fordiagnosis and treatment of enzyme-related conditions that are specificfor the subfamily of enzymes that the one of the present inventionbelongs to, particularly in cells and tissues that express the enzyme.Experimental data as provided in FIG. 1 indicates that the enzymes ofthe present invention are expressed in humans in the T cells from T cellleukemia, B. cells from Burkitt lymphoma, neuroblastoma cells, duodenaladenocarcinoma cell line, adenocarcinoma cell line, stomach and breastdetected by a virtual northern blot. In addition, PCR-based tissuescreening panels indicate expression in whole liver.

[0051] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the enzyme, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in the T cells from T cell leukemia, Bcells from Burkitt lymphoma, neuroblastoma cells, duodenaladenocarcinoma cell line, adenocarcinoma cell line, stomach, breast,whole liver. In an alternate embodiment, cell-based assays involverecombinant host cells expressing the enzyme protein.

[0052] The polypeptides can be used to identify compounds that modulateenzyme activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with the enzyme.Both the enzymes of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the enzyme. These compounds can befurther screened against a functional enzyme to determine the effect ofthe compound on the enzyme activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the enzyme to a desired degree.

[0053] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the enzyme protein and a molecule that normally interacts withthe enzyme protein, e.g. a substrate or a component of the signalpathway that the enzyme protein normally interacts (for example, anotherenzyme). Such assays typically include the steps of combining the enzymeprotein with a candidate compound under conditions that allow the enzymeprotein, or fragment, to interact with the target molecule, and todetect the formation of a complex between the protein and the target orto detect the biochemical consequence of the interaction with the enzymeprotein and the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

[0054] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0055] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantenzymes or appropriate fragments containing mutations that affect enzymefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

[0056] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) enzyme activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate enzyme activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the enzyme proteindependent signal cascade can be assayed.

[0057] Any of the biological or biochemical functions mediated by theenzyme can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the enzyme can be assayed.Experimental data as provided in FIG. 1 indicates that the enzymes ofthe present invention are expressed in humans in the T cells from T cellleukemia, B cells from Burkitt lymphoma, neuroblastoma cells, duodenaladenocarcinoma cell line, adenocarcinoma cell line, stomach and breastdetected by a virtual northern blot. In addition, PCR-based tissuescreening panels indicate expression in whole liver.

[0058] Binding and/or activating compounds can also be screened by usingchimeric enzyme proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native enzyme. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the enzyme is derived.

[0059] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the enzyme (e.g. binding partners and/or ligands).Thus, a compound is exposed to a enzyme polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble enzyme polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble enzyme polypeptide, itdecreases the amount of complex formed or activity from the enzymetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of the enzyme.Thus, the soluble polypeptide that competes with the target enzymeregion is designed to contain peptide sequences corresponding to theregion of interest.

[0060] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the enzyme protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0061] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of enzyme-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a enzyme-binding protein and a candidate compound are incubated inthe enzyme protein-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theenzyme protein target molecule, or which are reactive with enzymeprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

[0062] Agents that modulate one of the enzymes of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

[0063] Modulators of enzyme protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the enzyme pathway, by treating cells or tissuesthat express the enzyme. Experimental data as provided in FIG. 1indicates expression in humans in the T cells from T cell leukemia, Bcells from Burkitt lymphoma, neuroblastoma cells, duodenaladenocarcinoma cell line, adenocarcinoma cell line, stomach, breast,whole liver. These methods of treatment include the steps ofadministering a modulator of enzyme activity in a pharmaceuticalcomposition to a subject in need of such treatment, the modulator beingidentified as described herein.

[0064] In yet another aspect of the invention, the enzyme proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the enzyme and are involved in enzyme activity.Such enzyme-binding proteins are also likely to be involved in thepropagation of signals by the enzyme proteins or enzyme targets as, forexample, downstream elements of a enzyme-mediated signaling pathway.Alternatively, such enzyme-binding proteins are likely to be enzymeinhibitors.

[0065] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a enzyme proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aenzyme-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the enzyme protein.

[0066] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a enzyme-modulating agent, an antisense enzymenucleic acid molecule, a enzyme-specific antibody, or a enzyme-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

[0067] The enzyme proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in the T cells from T cell leukemia, Bcells from Burkitt lymphoma, neuroblastoma cells, duodenaladenocarcinoma cell line, adenocarcinoma cell line, stomach, breast,whole liver. The method involves contacting a biological sample with acompound capable of interacting with the enzyme protein such that theinteraction can be detected. Such an assay can be provided in a singledetection format or a multi-detection format such as an antibody chiparray.

[0068] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0069] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered enzyme activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0070] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0071] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Phannacol. Physiol. 23(10-11):983-985. (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the enzyme protein in which one ormore of the enzyme functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and enzyme activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0072] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the T cells from T cell leukemia, B. cells fromBurkitt lymphoma, neuroblastoma cells, duodenal adenocarcinoma cellline, adenocarcinoma cell line, stomach, breast, whole liver.Accordingly, methods for treatment include the use of the enzyme proteinor fragments.

[0073] Antibodies

[0074] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0075] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0076] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0077] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0078] Antibodies are preferably prepared from regions or discretefragments of the enzyme proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or enzyme/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

[0079] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0080] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0081] Antibody Uses

[0082] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat the enzymes of the present invention are expressed in humans in theT cells from T. cell leukemia, B. cells from Burkitt lymphoma,neuroblastoma cells, duodenal adenocarcinoma cell line, adenocarcinomacell line, stomach and breast detected by a virtual northern blot. Inaddition, PCR-based tissue screening panels indicate expression in wholeliver. Further, such antibodies can be used to detect protein in situ,in vitro, or in a cell lysate or supernatant in order to evaluate theabundance and pattern of expression. Also, such antibodies can be usedto assess abnormal tissue distribution or abnormal expression duringdevelopment or progression of a biological condition. Antibody detectionof circulating fragments of the full length protein can be used toidentify turnover.

[0083] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in humans in the T cells from T cell leukemia, Bcells from Burkitt lymphoma, neuroblastoma cells, duodenaladenocarcinoma cell line, adenocarcinoma cell line, stomach, breast,whole liver. If a disorder is characterized by a specific mutation inthe protein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant protein.

[0084] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin the T cells from T cell leukemia, B cells from Burkitt lymphoma,neuroblastoma cells, duodenal adenocarcinoma cell line, adenocarcinomacell line, stomach, breast, whole liver. The diagnostic uses can beapplied, not only in genetic testing, but also in monitoring a treatmentmodality. Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

[0085] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0086] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in the T cellsfrom T cell leukemia, B. cells from Burkitt lymphoma, neuroblastomacells, duodenal adenocarcinoma cell line, adenocarcinoma cell line,stomach, breast, whole liver. Thus, where a specific protein has beencorrelated with expression in a specific tissue, antibodies that arespecific for this protein can be used to identify a tissue type.

[0087] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the enzyme peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0088] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

[0089] Nucleic Acid Molecules

[0090] The present invention further provides isolated nucleic acidmolecules that encode a enzyme peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the enzyme peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0091] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0092] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0093] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0094] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO: 1, transcript sequence and SEQ ID NO:3, genomic sequence),or any nucleic acid molecule that encodes the protein provided in FIG.2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotidesequence when the nucleotide sequence is the complete nucleotidesequence of the nucleic acid molecule.

[0095] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0096] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID. NO:2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0097] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0098] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0099] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the enzyme peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0100] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0101] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the enzyme proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0102] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0103] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0104] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0105] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the map position was determined to be onchromosome 10 by ePCR.

[0106]FIG. 3 provides information on SNPs that have been found in thegene encoding the enzyme protein of the present invention. SNPs wereidentified at 57 different nucleotide positions in introns and regions5′ and 3′ of the ORF. Such SNPs in introns and outside the ORF mayaffect control/regulatory elements.

[0107] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0108] Nucleic Acid Molecule Uses

[0109] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.57 SNPs, including 8 indels, have been identified in the gene encodingthe enzyme protein provided by the present invention and are given inFIG. 3.

[0110] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0111] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0112] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0113] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0114] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. As indicated by the datapresented in FIG. 3, the map position was determined to be on chromosome10 by ePCR.

[0115] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0116] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0117] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0118] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0119] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0120] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that the enzymes of the present invention are expressed inhumans in the T cells from T cell leukemia, B cells from Burkittlymphoma, neuroblastoma cells, duodenal adenocarcinoma cell line,adenocarcinoma cell line, stomach and breast detected by a virtualnorthern blot. In addition, PCR-based tissue screening panels indicateexpression in whole liver. Accordingly, the probes can be used to detectthe presence of, or to determine levels of, a specific nucleic acidmolecule in cells, tissues, and in organisms. The nucleic acid whoselevel is determined can be DNA or RNA. Accordingly, probes correspondingto the peptides described herein can be used to assess expression and/orgene copy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease inenzyme protein expression relative to normal results.

[0121] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0122] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a enzyme protein, such as bymeasuring a level of a enzyme-encoding nucleic acid in a sample of cellsfrom a subject e.g., mRNA or genomic DNA, or determining if a enzymegene has been mutated. Experimental data as provided in FIG. 1 indicatesthat the enzymes of the present invention are expressed in humans in theT cells from T cell leukemia, B cells from Burkitt lymphoma,neuroblastoma cells, duodenal adenocarcinoma cell line, adenocarcinomacell line, stomach and breast detected by a virtual northern blot. Inaddition, PCR-based tissue screening panels indicate expression in wholeliver.

[0123] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate enzyme nucleic acid expression.

[0124] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the enzyme gene, particularly biological and pathologicalprocesses that are mediated by the enzyme in cells and tissues thatexpress it. Experimental data as provided in FIG. 1 indicates expressionin humans in the T cells from T cell leukemia, B cells from Burkittlymphoma, neuroblastoma cells, duodenal adenocarcinoma cell line,adenocarcinoma cell line, stomach, breast, whole liver. The methodtypically includes assaying the ability of the compound to modulate theexpression of the enzyme nucleic acid and thus identifying a compoundthat can be used to treat a disorder characterized by undesired enzymenucleic acid expression. The assays can be performed in cell-based andcell-free systems. Cell-based assays include cells naturally expressingthe enzyme nucleic acid or recombinant cells genetically engineered toexpress specific nucleic acid sequences.

[0125] The assay for enzyme nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the enzyme proteinsignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

[0126] Thus, modulators of enzyme gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of enzyme mRNA inthe presence of the candidate compound is compared to the level ofexpression of enzyme mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

[0127] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate enzyme nucleic acid expressionin cells and tissues that express the enzyme. Experimental data asprovided in FIG. 1 indicates that the enzymes of the present inventionare expressed in humans in the T cells from T cell leukemia, B cellsfrom Burkitt lymphoma, neuroblastoma cells, duodenal adenocarcinoma cellline, adenocarcinoma cell line, stomach and breast detected by a virtualnorthern blot. In addition, PCR-based tissue screening panels indicateexpression in whole liver. Modulation includes both up-regulation (i.e.activation or agonization) or down-regulation (suppression orantagonization) or nucleic acid expression.

[0128] Alternatively, a modulator for enzyme nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits theenzyme nucleic acid expression in the cells and tissues that express theprotein. Experimental data as provided in FIG. 1 indicates expression inhumans in the T cells from T cell leukemia, B cells from Burkittlymphoma, neuroblastoma cells, duodenal adenocarcinoma cell line,adenocarcinoma cell line, stomach, breast, whole liver.

[0129] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe enzyme gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0130] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in enzyme nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in enzyme genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the enzyme gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the enzyme gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a enzyme protein.

[0131] Individuals carrying mutations in the enzyme gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the enzymeprotein of the present invention. SNPs were identified at 57 differentnucleotide positions in introns and regions 5′ and 3′ of the ORF. SuchSNPs in introns and outside the ORF may affect control/regulatoryelements. As indicated by the data presented in FIG. 3, the map positionwas determined to be on chromosome 10 by ePCR. Genomic DNA can beanalyzed directly or can be amplified by using PCR prior to analysis.RNA or cDNA can be used in the same way. In some uses, detection of themutation involves the use of a probe/primer in a polymerase chainreaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), suchas anchor PCR or RACE PCR, or, alternatively, in a ligation chainreaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080(1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter ofwhich can be particularly useful for detecting point mutations in thegene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

[0132] Alternatively, mutations in a enzyme gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0133] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0134] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant enzyme gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0135] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0136] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the enzyme gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the enzymeprotein of the present invention. SNPs were identified at 57 differentnucleotide positions in introns and regions 5′ and 3′ of the ORF. SuchSNPs in introns and outside the ORF may affect control/regulatoryelements.

[0137] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0138] The nucleic acid molecules are thus useful as antisenseconstructs to control enzyme gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of enzyme protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into enzyme protein.

[0139] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of enzyme nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired enzyme nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the enzyme protein, such as substratebinding.

[0140] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in enzyme geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired enzymeprotein to treat the individual.

[0141] The invention also encompasses kits for detecting the presence ofa enzyme nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the enzymes of the present inventionare expressed in humans in the T cells from T cell leukemia, B cellsfrom Burkitt lymphoma, neuroblastoma cells, duodenal adenocarcinoma cellline, adenocarcinoma cell line, stomach and breast detected by a virtualnorthern blot. In addition, PCR-based tissue screening panels indicateexpression in whole liver. For example, the kit can comprise reagentssuch as a labeled or labelable nucleic acid or agent capable ofdetecting enzyme nucleic acid in a biological sample; means fordetermining the amount of enzyme nucleic acid in the sample; and meansfor comparing the amount of enzyme nucleic acid in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectenzyme protein mRNA or DNA.

[0142] Nucleic Acid Arrays

[0143] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ IDNOS:1 and 3).

[0144] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0145] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0146] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0147] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0148] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0149] Using such arrays, the present invention provides methods toidentify the expression of the enzyme proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of the enzymegene of the present invention. FIG. 3 provides information on SNPs thathave been found in the gene encoding the enzyme protein of the presentinvention. SNPs were identified at 57 different nucleotide positions inintrons and regions 5′ and 3′ of the ORF. Such SNPs in introns andoutside the ORF may affect control/regulatory elements.

[0150] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0151] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0152] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0153] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0154] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified enzyme gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

[0155] Vectors/Host Cells

[0156] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0157] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0158] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0159] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0160] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0161] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0162] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0163] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0164] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0165] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0166] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0167] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enteroenzyme. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0168] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0169] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES² (InvitrogenCorporation, San Diego, Calif.).

[0170] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0171] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0172] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning:. ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0173] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0174] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0175] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0176] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0177] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication Will occur in host cellsproviding functions that complement the defects.

[0178] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0179] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0180] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such asenzymes, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0181] Where the peptide is not secreted into the medium, which istypically the case with enzymes, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0182] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0183] Uses of Vectors and Host Cells

[0184] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga enzyme protein or peptide that can be further purified to producedesired amounts of enzyme protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0185] Host cells are also useful for conducting cell-based assaysinvolving the enzyme protein or enzyme protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native enzyme protein is useful forassaying compounds that stimulate or inhibit enzyme protein function.

[0186] Host cells are also useful for identifying enzyme protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantenzyme protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native enzyme protein.

[0187] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a enzyme proteinand identifying and evaluating modulators of enzyme protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0188] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the enzyme proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0189] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the enzyme protein to particularcells.

[0190] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0191] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0192] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0193] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo'and that could effect substratebinding, enzyme protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo enzymeprotein function, including substrate interaction, the effect ofspecific mutant enzyme proteins on enzyme protein function and substrateinteraction, and the effect of chimeric enzyme proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more enzyme proteinfunctions.

[0194] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 4 1 3433 DNA Homo sapiens 1 ctccgcacgc ccctgccccg tcgcctcccgggagcggctg cagtccccgt tggctgcaga 60 cgggcagaca cccgaagtgt ccgcgccggcagccggaccg caagcgatga atatcgaacc 120 gcgctcccgg gcctggccga agcgcttcctgtgccaggtt tccggctccg ggtcccggga 180 ggaggatcgc gggccattat gcggagccgccgccctgccg cgactttcag actccgacca 240 tggcctcgcg ctggtggcgg aggcggcgcggctgctcctg gaagccggcg gcgcggagcc 300 ccgggcccgg ctcccccggc cgtgcgggaccgttggggcc gatcgccgct gccgatattc 360 cgcgcgcagg ttcataggcg gaagggacttgacttgtctc agatacccta tattaatctt 420 gtgaagcatt taacatctgc ctgtccaaatgtatgtcgta tatcacggtt tcatcacaca 480 accccagaca gtaaaacaca cagtggtgaaaaatacaccg atcctttcaa actcggttgg 540 agagacttga aaggtctgta tgaggacattagaaaggaac tgcttatatc aacatcagaa 600 cttaaggaaa tgtctgagta ctactttgatgggaaaggga aagcctttcg accaattatt 660 gtggcgctaa tggcccgagc atgcaatattcatcataaca actcccgaca tgtgcaagcc 720 agccagcgcg ccatagcctt aattgcagaaatgatccaca ctgctagtct ggttcacgat 780 gacgttattg acgatgcaag ttctcgaagaggaaaacaca cagttaataa gatctggggt 840 gaaaagaagg ctgttcttgc tggagatttaattctttctg cagcatctat agctctggca 900 cgaattggaa atacaactgt tatatctattttaacccaag ttattgaaga tttggtgcgt 960 ggtgaatttc ttcagctcgg gtcaaaagaaaatgagaatg aaagatttgc acactacctt 1020 gagaagacat tcaagaagac cgccagcctgatagccaaca gttgtaaagc agtctctgtt 1080 ctaggatgtc ccgacccagt ggtgcatgagatcgcctatc agtacggaaa aaatgtagga 1140 atagcttttc agctaataga tgatgtattggacttcacct cgtgttctga ccagatgggc 1200 aaaccaacat cagctgatct gaagctcgggttagccactg gtcctgtcct gtttgcctgt 1260 cagcaggtag gttttacaaa ctccctttgacacatcactg catagcccca cagaactgat 1320 gtcccgcggc acagctgatg ggaagattgcataaaggaat agatgggaag gcattcagat 1380 aagagatcac aggtctgcat ttgatcctggctgagtgaga tgttggggct ggtcatttca 1440 ccttgctaag actgtttcct tatctgtaaaattgagaaga tcacctttct cccagggtgg 1500 ttgtgaggat tagctaagat cctatttgagatctttgtgt cttgtggtgt gccataggca 1560 tttgaggtag catcgtgatt atttccatatattttggcca ctggcaaagt gaacggtttc 1620 taagtcttga ttataggact ggactttggtggtcctcaga gccccttaaa aggcatagga 1680 agcatcaagg gcctccaagc ataagaaattctccggttct agaagtttaa tgagactctg 1740 ctgctctgag agaggcttta gaacctcggccattgcctca aaatgtcagg aagtcagtgg 1800 agtgcagtag acccacatag ttccttctttctccggattg agggactgag tcccccttaa 1860 tgtgaatgaa aggcttagga agcttcaaagatgttccctc gactgacaaa gcagacattc 1920 tcacagcctc ctccagaccc tgccacatggcttgtggctg tactgaatgt tacttgaaat 1980 aagtgagaca ttagctggtg ttggaacatctcgttaatag attttcatct tagtagtatt 2040 taatttgtta tgttgcaaag cagtaagatgttcatcaccg tgccatgaaa ttcaacatta 2100 gctctttggt gtaaaattat agtaacttttggtctttcag agattttgcc tctattctgt 2160 cttcacgttt acaaaggtca gtcatgtcctccataaaatt cagtgattcc actgtgatac 2220 agaaaccacg gcccttgctt ttggtgggtttctgattgga gagaggaaag gtcatctttc 2280 acccactatc tagcatagcc attggcagcatgattcttcc caggggaggc tgacgttctg 2340 ggtggctgga ccaggctact ttggcagcttgctaaggcta tgaatggaga tgttggggta 2400 ctcggtagga acacccgccc tcattattacaaggcttcca tcctctcaaa ctttggaggc 2460 tgaggtaaga agtgaaaggt atgctgtaaataggtcctct ctcccaatga ggcttacttg 2520 ccagcccaaa atcaaagagt ataatacatgtgcccagttt tgacaaaaat ttataaaacc 2580 tccttttgta cattaaggca agagtgaggaacatttgagc catgtaggtg ttatgctggg 2640 gattagaaaa atgaggcact ggctaccagtaacctatata actgcgaaca ttacttctca 2700 gatacttgtt agtaaacatg agtgaaggaaagcaagatgg actgagtgtg ctgaaatcca 2760 gctagcttgg taaagattcc tttacctaggctcagattat caggataaaa ggaaaaagcc 2820 tttttccctg gagaagtcta tgagaaagttttggttgctc tatttgtaaa aatcttcaaa 2880 ttgttaagta cttgttatga accccaggatactaagttac cggttgagtc ctacttaaac 2940 cttaaggtga ctgggtgaga ggaggctggcctcttcggac tgtgtttcac tctgaatata 3000 tttcagaaga aactaactta ctttcccctacacacacaaa ggagtaatgg ctatctctgc 3060 tttcatatat agtgggggaa aggggaaatggacctctgca tagtatctgt cagtaatcta 3120 caagagactg aaaaatgctg gttaggcggtggctcatgcc tgtaatccca gcactttggg 3180 aggctgaggc agttggatta tgaggtcaggagttcaagac cagcctgacc aatatggtgg 3240 aaaccccgtc tctactaaaa atacaaaaattagccgggcc tggtggtgca tgcctgtaat 3300 cccagctact cgggaggcca aggaaggagaatccttgaac ctgggaggca gaggttgcag 3360 tgagccgaga ctgcactcca gcctgggtgacagagtgaga ctccgtctca aaaaaaaaaa 3420 aaaaaaaaaa aaa 3433 2 363 PRT Homosapiens 2 Met Arg Ser Arg Arg Pro Ala Ala Thr Phe Arg Leu Arg Pro TrpPro 1 5 10 15 Arg Ala Gly Gly Gly Gly Gly Ala Ala Ala Pro Gly Ser ArgArg Arg 20 25 30 Gly Ala Pro Gly Pro Ala Pro Pro Ala Val Arg Asp Arg TrpGly Arg 35 40 45 Ser Pro Leu Pro Ile Phe Arg Ala Gln Val His Arg Arg LysGly Leu 50 55 60 Asp Leu Ser Gln Ile Pro Tyr Ile Asn Leu Val Lys His LeuThr Ser 65 70 75 80 Ala Cys Pro Asn Val Cys Arg Ile Ser Arg Phe His HisThr Thr Pro 85 90 95 Asp Ser Lys Thr His Ser Gly Glu Lys Tyr Thr Asp ProPhe Lys Leu 100 105 110 Gly Trp Arg Asp Leu Lys Gly Leu Tyr Glu Asp IleArg Lys Glu Leu 115 120 125 Leu Ile Ser Thr Ser Glu Leu Lys Glu Met SerGlu Tyr Tyr Phe Asp 130 135 140 Gly Lys Gly Lys Ala Phe Arg Pro Ile IleVal Ala Leu Met Ala Arg 145 150 155 160 Ala Cys Asn Ile His His Asn AsnSer Arg His Val Gln Ala Ser Gln 165 170 175 Arg Ala Ile Ala Leu Ile AlaGlu Met Ile His Thr Ala Ser Leu Val 180 185 190 His Asp Asp Val Ile AspAsp Ala Ser Ser Arg Arg Gly Lys His Thr 195 200 205 Val Asn Lys Ile TrpGly Glu Lys Lys Ala Val Leu Ala Gly Asp Leu 210 215 220 Ile Leu Ser AlaAla Ser Ile Ala Leu Ala Arg Ile Gly Asn Thr Thr 225 230 235 240 Val IleSer Ile Leu Thr Gln Val Ile Glu Asp Leu Val Arg Gly Glu 245 250 255 PheLeu Gln Leu Gly Ser Lys Glu Asn Glu Asn Glu Arg Phe Ala His 260 265 270Tyr Leu Glu Lys Thr Phe Lys Lys Thr Ala Ser Leu Ile Ala Asn Ser 275 280285 Cys Lys Ala Val Ser Val Leu Gly Cys Pro Asp Pro Val Val His Glu 290295 300 Ile Ala Tyr Gln Tyr Gly Lys Asn Val Gly Ile Ala Phe Gln Leu Ile305 310 315 320 Asp Asp Val Leu Asp Phe Thr Ser Cys Ser Asp Gln Met GlyLys Pro 325 330 335 Thr Ser Ala Asp Leu Lys Leu Gly Leu Ala Thr Gly ProVal Leu Phe 340 345 350 Ala Cys Gln Gln Val Gly Phe Thr Asn Ser Leu 355360 3 39982 DNA Homo sapiens 3 catcaatatg gcaagaaaat gtcttggagggtacctaaca ctccaacctc aaaacgacaa 60 ccttgatcgc tttcagttac acataggccattttgatcca tctgtactgg ctcttttcat 120 ttgtctaagg gcagcaaaat agactaaagttgttttcaga ctgcacaaga gaagtctgtt 180 ctttagcact tacctgcata actcagatagtgtttttaac ttcctcaact catgcttggc 240 ctagttgggt caaaagaact acttgtgaattccgtatgtc caattggtag gttactgttt 300 tttctcaaga gtttagctat acagtaatgttggaagtctt gaggaacttc agctgctgtc 360 cctacttagc cttgcaatac tacggcatcttttcttcttt ttcttttttt taaagacgga 420 gtctcacttt gtcacctagg ccgaagtgcagtggcacaat ctcggctcac tgcaacctct 480 gcctcccagg ttcaagtgat tctcttgcctcagcctccta agtagttggg attacagaac 540 atgcaccacc atgcccaact aatttttaaatatttagtag agacgggttt caccatgttg 600 gccaggctgg tctcaaactc ctgacctcaagtgatccacc tgcctcggcc tcccacagtg 660 ctgggattac aggctgagcc actgtgcccccgacctacag catcttttta agagcaatgt 720 tttctgtttt aatgtggcca agcaatctctaaacgaacat ggtctgacaa aattgtgtag 780 tgacatactt gacaactggg tgagcttgagtgtgatactt atttgaccat acggtatttc 840 ctctatatgt ttccttatct ttatatacaaaaaagggcaa atgataatgt cttagtcata 900 ggatcaagta caacccaagt gcttactgctacactctact tggcaaattc caccttatga 960 atgtttctta atatataaac ctaatctaaaccaagataag ctacaggtgc atattaacaa 1020 tctttactca taaagttttg catatttttacatactattc ttcaagatgg aaaaaacacc 1080 atcaaactgg cttattttca tagatgtctttatatagtct atgatgtctt cattccataa 1140 aaaatccaca cgattaaata tgaaattaatgtacccagct atgaggctac cgcattcttt 1200 aacccaagaa caagaaaata catgaatcagagaactgcaa ttacctactt gctggtctat 1260 ttcctcatta gagtgaattt tttgaggactaaattgcatt tctctatctc taatgcttac 1320 cccctatctc actggctctg tgtgtttgagtgcatgcgct gaatgtttat atcacttaat 1380 ttctgttcgg aacacgctgt ctcttagcaccagtagatct ggccttagag ctaaggaaag 1440 aaggcctttc taacccataa ggggtactaatcctgtacag actccaatat ctcccgggct 1500 gggtacacgc aggttcacac ccaggtgtgagcaagattgc caaactatac ctataaggcg 1560 tttggaggag agagtccaaa agtcacgagacccggctaat ttagctacta tttcacaaca 1620 accctgatcg tggtcggact tgtttctaggaagatgcgat gggctggaac ttccttcaag 1680 aacagattgc aaaaggcagc cgaccagggcgtgtcaggtc actgccaagg gaagcagcag 1740 cggaaaccgt cgcctccggc ttaggctgcggagcgggaac cccattctct gagttatgtt 1800 tctgcctaaa cacgcagaac aaaagagttcgtggctgcaa gggtggcgcg cggtagtatt 1860 tcttgctaat aaaaggtccc cacagggacatttttgctat gactgcaggc gtggccagtg 1920 cccctgcaca gcttccggga agaggtggggataggaggct cgccccgggg aaggtcacgc 1980 tcggggtccc cagaccaggt ctccgcacgcccctgccccg tcgcctcccg ggagcggctg 2040 cagtccccgt tggctgcaga cgggcagacacccgaagtgt ccgcgccggc agccggaccg 2100 caagcgagga agagcgaacc gcgctcccgggcctggccga agcgcttcct gtgccaggtt 2160 tccggctccg ggtcccggga ggaggatcgcgggccagagg cggagccgcc gccctgccgc 2220 gactttcaga ctccgaccat ggcctcgcgctggtggcggt ggcggcgcgg ctgctcctgg 2280 aagccggcgg cgcggagccc cgggcccggctcccccggcc gtgcgggacc gttggggccg 2340 agcgccgctg ccgaagtccg cgcgcaggtgaggttgggag gcgcgcgccc ggcggggctc 2400 agaggtcacg gctccaatga cagcagtgggcggaatgaat gggagcgggg agcacgtgcg 2460 tcgcgacgcg ggggcgcgcg gggtagctccggggtagctc cggggtggga ctccggagct 2520 gaggggtgct cgcggtggga cggagccgcgcgttggactg aagtaggggc gccctacacg 2580 gggttgcaga aagcggtgtc ctggggaccccgggagcgtt ttggggggtg gagaaagcgg 2640 tgggatccgt gtcctggggg aagcgcaggagcgatcagat tgaccgctgc tatatggaca 2700 aggttaatta agcctggaga gcgactgcagctatttactt tggagttaga ggaagacaga 2760 tcttgcctga aattttagag ccaaagtgctcagagctctc tccgagaaac ggaggatcga 2820 atcggcctcg gaatagtccg aaaaggtcttgtggagaatg cagggctggt gctagggtgg 2880 accggacctt agttgtccag cagtggcttcgggaggagag cacacctgga tggggctgga 2940 atgacactgg aaggagaaag gcccgaggccgtcaggagga gagaaaccct ggagtcatct 3000 aaagtcagaa tctcagtgat agcccattctttcagggtct cctctgctgc ctttgggttc 3060 cgatctcctt tttctccctt tctggtcagtttagcccctg taaatggaca ctgtgagccc 3120 ttctgcactt tgtaatgatt ctcagtgtgccatggggtag gcccatgcta ttagcataac 3180 cgtgcaaaat ctatggactc actgttacttaagtttattt gaatttagta ctcttttaat 3240 tcagatttgg tatactttat gtgtccgtgttctcatcaaa catcatttcc cagtatgaaa 3300 tatataacac ttataaatat ctactttgtaccaaagtaga tgcgtatcac aaattatcca 3360 aaacaaggct tgggccaggc acagtggctcacacctgtaa tcccagcact ttgggaggct 3420 gaggtgggag aatcggttga ggccaggagtttgggaccag cctaggcaac atagcaagac 3480 tctgtctcta taaaaaatag aaaaaattcaccaggtgggg tggtgcatgc ctgtagttct 3540 acctactcag gagcctgagg gaggaggattgttcgatccc aggagttcaa gattacagtg 3600 agctgtttat tgtgatagtg ccagtgcactccagcctggg caacagtgtg agaccctatc 3660 tctaaaaaat ctaaagcaaa aaagacgtttggagaccaag tggcagctgg gcaactagac 3720 aatgctagag agtaaagaga acagaatatggggggtgagg ggcacgtggg agaagaggct 3780 tagaggagga aggtaggctt tgctgtgatccatttcagga ctgttctctc cagacttcag 3840 atatttctag ttccacagaa gaagccagtatttgaagaga tgggtgcatc caccctctta 3900 tgccactaca tgcttcttct tcttcttctttttttttttt ttttgacaca gagtctcact 3960 ctgtcgccca ggctggagtg cagtggcggaatctcggctc actgcaaact ccacctccca 4020 ggttcaagcg attctcctgc tgcagactcccaagtagctg ggattacagg cgtacaccac 4080 gacccccagc taatttttgt atttttagtagagacagcat ttcaccatgt tgcccaggct 4140 ggtctggaac tcctgacctc aagtgatccaccccctcctc ggcctcccaa agtgcgggga 4200 ttacaggtgt gagccaccac gcctggcctgacacttcttt ttaaaaaagt ttcttgccca 4260 ccaagcaacc ttagccaggg aaagtgtgcttttctggaga gaatatgctt tatggcctcc 4320 ccgtctgcat gacgacaagg aggaattctgtggactcatg atgccaagat catgtatttc 4380 tgatgtgtgt gtcagtgatg cctttcactatacgttaata tgttagcatt ctggtgtctg 4440 cctcaggcat gtgtattttg gttacttcaagtctcttagg gcctgatagt ccttggggac 4500 acagaactac aataactaag ccacagggacaaagagcggc aataattaag ccacgtcttg 4560 ctctccacca gagctctact acctcctaatggttgtacat tcttctgact tcactagtag 4620 tgtgaacaaa tattgtaata caataaaaaccagtgtgcat gtgatcgtgc caaatcactt 4680 ctcaatatat ctttgatttt tttttttaatttattttttt agagacgggg actttttgtg 4740 ttggccaggt tggtcttgaa ctcttggactcaagccattc ccccctcccc cccaccccgc 4800 ttgaggattg gcacacggcc atgtatttgattcttaccca gcactttctc tttagctgag 4860 cgtggtggct cacgcctgta atcccaacagtttgggaggc cgaggcagga gaatcccttt 4920 agctcaggag ttcgagacca gcctgggccacttagtgaga cctcctgata cggtttggct 4980 ctgtatgccc acccacatct atctcgaactgtaatcccca attacatttg gggaccaggg 5040 agggtcaagg gagggaccag gtggaggtaattggatcatg ggggtggatt cccccatgct 5100 gttctcatga tagggagttc tcacaagatctgctggtttt ataagtgttt ggtagttcct 5160 cctgtgttca ttctgtctcc tgccgccttgtgaagaaggt gcttgtttct ccttctcctt 5220 cttccaccat gattctaagt ttctgaggcctccccagcca ttcagaactg tgagtcaatt 5280 aaacctcttt cctataatta taaattgcccagtcatgggt aatttcttta tagcagtgtg 5340 agaatggacc aatacagata attggtaccaaagtagttgg cattgatata agatacctga 5400 gaatgtggaa attactttgg aactgggtaacaagcagagg ttggaatagt ttggagggct 5460 cagaagaaca aaggaagatg taggaaagtttggaaattcc tagagtcttg ttgaatggct 5520 ttgaccaaaa tgctgaaagt gatatggatgatgaagtcca ggctgaggtg gtctcagatg 5580 gagatgagga gcttcctggg aactgggcaaaggtcactct tgctatgctt tagcaaagag 5640 actggtggca ttttgcccca ccctagagatctgtggaact ttgagcctga gagagatgat 5700 ctgaaattgg aacttaaatt taaaggggaagcagagcata aacgtttgga aaatttgcag 5760 cctgaagatg tgatagaaaa gaattttctggggaggaatt caagctggct gcagacattt 5820 gcataagtaa caaggagccg aatgttaatagccaagagaa tggggaaaat gtctccagag 5880 catgtcagag accttctctg cagcccctcccatcacaggc caggaggcct aggagggaaa 5940 aatggattca tgggctgggc ccagggccctgctgctctgt gcaagagcag ccttgggact 6000 tggtgccctg cttcccagcc gtagctaaaaggggccaagg tacagcttgg gccattgctt 6060 cagagggtat aagccccaag ccttggtggcttccatgtgc tgctgagcct gcaaggtgca 6120 cagaagacaa caatttggga acttctgcctagatttcaga ggatgtatgg aaatgcctca 6180 atgcccaggc agaagtctgc tgaaggggggagaaccctca tggagaacct ctgttaggac 6240 agtgcagaaa ggaaatgtgg ggttgcagcccccacacgga gtccctgctg gggcactgcc 6300 taatggagct gtgaaaggag ggccaccgtgctctagaccc cagaatggta ggtccaccaa 6360 cagcttccac cgtgcacctg gaaaagctgcaggcactcaa tgccagccca tgagaatagc 6420 agtgggggtt gaaccctgca aagccacagaggcagagcca cccaagttct tgggagccca 6480 ccctttgcat cagtgtgacc tagatgtgagacatggagtc aaagatcatt ttggagcttt 6540 aagtaatgac tgccctgctg ggtttggacttgcatgaggc ctgtaagccc cttcgttttg 6600 gccaatttct accatttgga atgggaacatttaaccaatg cctgtacccc cattgtatct 6660 tggatgtaac taacttgttt ttgattttacaggttcatag gcggaaggga cttgacttgt 6720 ctcaggtaag actttggact tggacttttgagttaatgct agaatgagtt aagacttttg 6780 aggactgttc ggaaggaatg attggttttgaaaaatgagg atatgagatt tgggaggagc 6840 aaggggcaga acgatatggt tggttctgtgtctctaccca aatctcatct caaattttaa 6900 tccccatgtg tcaaaggagg gcctatgtggaggtaattgg atcctggggg cagattcccc 6960 catgctgttc ttgttatagc gaattcttatgagatctgat ggttttataa gtgattggta 7020 gttccttctg tgttcattct ccttcctgctgcctagtgaa gaaggtgcct tgctttccct 7080 tttccttctg ccatgattgt aagtttcttgaggcctccct agccgtttgg aactgtgagt 7140 cagttacacc tctttctttt ataaattacccagtcttggg tatttcttta tagcaatgtg 7200 aaaacgtact aatacacctc gtttctaaaaatcagctacg cattgtggca tgttcctgta 7260 gtcccagcta ctcagtaggc tgaggtgggaagatagcttg agcccaggag attgaggctg 7320 cagtgagcta tgattgcacc actgcactccagcctggaaa aataaaataa aagtaaattc 7380 atgagaggca aggttgactt tcagagagtgtgtgagggaa aggtaagaaa caaaatcgga 7440 attagaaaat gactgttgcc cctctcttttcattcttcac tgcttttctg agcacctctc 7500 acctcccttt gaaaaactgc tctaaagtacttttaaagca gaaaatagta gcttgattga 7560 atagatactg tattaaatgt atattgtgtaccaggctctg agctaagagc tttacatctg 7620 ctctctaaat taatctccac ctctctccatgagtaggtat tatcagaaag acatcacaag 7680 gtaacacaaa tgttgagatt tgaaccgagatctgtcttcc aaagtccatg ctactaataa 7740 ttgttaggcc acttggtggt aaaaagcgtcccctgtatta attagctgtg taattttaat 7800 ccttcaaccc agattagggg tctataactgaataaccaaa aagttattca gttagaatag 7860 ttcagttagt tattaactaa tcatagttatttggtcaagc gcagtgactc acgtctgtaa 7920 ttccagcact tggggaggct gtggcaggaggatttctaat aatagtaact attactagag 7980 acctgataat aattattaag tctcttactgaagaagtttt actcttccat taagatgccc 8040 ttatttataa gtattataat aaaatactgaactcatatga attaattacc catgtagact 8100 gaaagacata ggttgtcata taatggttgtcatataaagc atgaaggagg attgtcactt 8160 tgttttcttg ttgcactgta attgcctgggatatcatgaa agttcttact ggtttaatga 8220 atatgtaatg ctgtggggga aagaatgaaactcaggccaa ctactttgac tggtgtctgg 8280 caaagtcaac tttcattgcc attttaatatggcgccgggc gcggtggctc acgcctgtaa 8340 tcccagcact ttgggaggcc aaggcgggcggatcatgagg tcaggagatc gagaccatcc 8400 tggctaacac ggtgaaaccc catctctactaaaaatacaa aaaattagcc aggcgaggtg 8460 gtgggcgcct gtagtcccag ctactcaggaggctgagaca ggagaatggc gtgaaccctg 8520 gggggcggag cctgcagtga gccgagatagcgccactgca ctccagcctg gacgacagaa 8580 cgagactccg tctcacaaaa aaaaaaaaaaaaaaatatgg ctagagcaat acatgttcat 8640 tagagaagaa tcagaaaata tctaccaaaagaaggaataa aacaaaacac ccccacaatc 8700 cgattcattc cagaataact acttttaacaccctggtttg tgtgctttca aactctgttc 8760 tcccatttcc aaaggatgtt gaggaacacgaacttaaaat agacagtgtt tgtataaata 8820 taaattcctt ccttgctatt gaaatcttttttcctctgac caatcttagt taaatggaag 8880 gaaattgttt taaaactaat tttcaggtctggaaagatag atatgaatat ggaaatgact 8940 ttctttgggg gaagggatgg actgaagatggttattattt tctccttttt tcttatttat 9000 gttttctgca atgacaagta ttttgtaataacaaaaggaa actttaaaat tattcttcct 9060 ctaaagtttg caatctacct gtaaatgggtgattagaaga taaagattat aattatgggg 9120 gttttatcat ccaatgttac agtttttcaggaatattctt acaagtttct tgacattttt 9180 attttataga taccctatat taatcttgtgaagcatttaa catctgcctg tccaaatgta 9240 tgtcgtatat cacggtaagt ttacagtccatactgcaact actaaaatta tccattttta 9300 aatttattat tgtttaaaaa attttttttgtagcgatggg gagctcactg tgttgcccag 9360 gctggtcttg aactgctggc ctcaagctatcctcccaccc tcaggttccc aaagtgctgg 9420 gattgcagac ataagcaccc ggcctaaaattattaattat attgcctgta aatttctatt 9480 ctaaattgta gatctctgcc tattcaaaaaacaggaatat aataaagttt gagctcaacc 9540 cagagcacaa tgaacatagt ttagtttttctttgattttg tgggttctca aggccctatt 9600 tataaaagtg atctattgat ctgtcatttagcaagaatag aattctgtat gtttttccaa 9660 attataatga ccttttcaga ttcatgattaatttctagca aatatttggg ctgaattttc 9720 cgtatctgag tctactaaat atatatgtatataaaactta cttgaaaatg aagtcatgtg 9780 catttttgca tgtcccaggt ttcatcacacaaccccagac agtaaaacac acagtggtga 9840 aaaatacacc gatcctttca aactcggttggagagacttg aaaggtctgt atgaggacat 9900 tagaaaggtg agttttttat tctgctgtgatgtaatgttt tagcttacca aaacttacta 9960 aaattttatt ttatttttta ttcttataattattattatt ttttgagctg gagtctcact 10020 ctgttgccca ggcttgagtg cagcggtgcaatctaagctc actgcaaccc ttgcctccca 10080 ggttcatgca attctcctgc ctcagcctcctgagtagctg agattatagg catgcgccat 10140 cacacctggg taatttttgt atttttagtgaagacggggt tttgctatgt tggccaggct 10200 ggtcttgaac tcctgacctc aggtgacctacccgccttgg cctcccaaag tgctgggatt 10260 acaaaaaaac agtgcaggtt ttcagatctgtacaatgcag tttgcaatct tgattcacgt 10320 atggtcaagt ttcaaatgtt tcttgagaagaatacatatg acccagtgcc aggcaatatg 10380 aagaatgcaa tatgtattta tgtccagaaagaggttatgg cagggttggg aacctgaagg 10440 aaaaaaatgg tccaggtagc tcatgcttgtccaatgagtt atcccacctt tcctcttaaa 10500 acccaccacc tcccagtgac tccagctgtctcttccttat ctaattctga atgtcattgc 10560 cagtgtgccc caaatatggc tttcttcattcaactctttt tcctgaaaac tttcagtagt 10620 tcccaacctc acgtggttgg gcaccgtggctcgtgcctgt aatcccacca atttaggagg 10680 ccaaagcaag aggactgctt aagcccaggagtttgaggct gcagtgagcc atgatcccgc 10740 cattgcactc cagcctgagt gagagagcaagacatttttt ctgtctttag aaaaaaattg 10800 gctgggcacg gtggatcaca cctataatcctagcactttg agaggacacc acttcctgtt 10860 tcacagacag taggggctgt ggaggaagaactccttcagc tcctgctctg tcgcaattgg 10920 ccagcttgcc tgcgccttct ctggcgattgcctgctctcc tcccacccgt ggaagtcatg 10980 tcccttccct ctctaggggc agtcccttagccaacctccc tagtttctta ggaactcccc 11040 cagacatggc ctctccctct gtctgcaaactttcattggc atggtcttcc atatccattg 11100 ggtgttcagt ttcttcacct gcatttttaaaaggcccatc cactgaccca gtgcttcact 11160 ccctcccttc cctcacagta tcccctgccctctgccttcc tccagtgcct tcatgttccc 11220 tccactattg cactggggcc ccctttccagcctctcctct gaagctggtt ttgcttgggt 11280 cactgatagc ttccctgtgc taaatccaatggatgtgtta ccagcagcaa atttgtacag 11340 gtctgcagca acctcaattc ttgcctcctcagaagaaaga attcgactga ggggcctaag 11400 gcagaaggag agacggaggc aagttttagagcaggagaga aagtttatta ttaagtgtag 11460 agtaggaagg gaaggaagta aagtacacttgtaagagggc caagctggtg acctgagaga 11520 aagtgtggtt tgaccttgga atttgggttttagatgttgg catacttcca gggacttgca 11580 tcccatctcc ccggtttctt cccttggggtgggctgcccg catgcgcagt ggcccgccag 11640 tgttggggag gggagcatgc tcagtgtgtttaccggagtt ttgcgcatgc tcacgtgagg 11700 cattcttccc ataccagtcc cagttttcctagaaggacat acaccaatta agctctgcca 11760 ttttgcctct tagtgtgcat ggttgagccgactcacccaa ctcccgagat cttattggga 11820 agctgatcac cagtttcagg tttttgtatctattgagaga ctgccgtccc ttggcaccag 11880 ctgtgaccaa ttagtatttt agcgacacagttaacaactg cttgaccatc acgtgatggt 11940 cgccttcctg ttgtgggtcg gggagccctctcctgccctg ctcatgcctg actagctatc 12000 tactgtaacg gacacttcct ggttttctgaccattgggca ctattgactg taacccctcc 12060 ttgaaacatg gttcccttca ctctgcactttgttccccct ccttatcctg aggcttttct 12120 tcggttcatc acctccagct cacttcctttaattttgcca ttccctgggg ttccgtgggc 12180 cccctcctct ctctcttcct tatatatctgtgtccatact tacgtctctc aaataatatc 12240 actactccag gttgaatcac atctcccacacccaggtagc taactactca ccgggaacgc 12300 catggggcgg ccatgacggt ctcaggcttaactcagctga gactctactt ttcccccacg 12360 agttgtttgt tctgctgttt tcctgttccttgggaacctg ggaagaaccc aggacttgtc 12420 cctctccttc ccctctatgt ccttctagtctgcctcttgt cagtccccac tgccccaggc 12480 caggcaattc catgcccctg gtcctttcattcgtgctctt ctcctccctg tctcagcttc 12540 cgaggtgaca gcttacctgg gtgacagcaccacacccatc acactgtgtt gtgtcaacgg 12600 cagtgatcgt tttacttctc cccatctagaccctgagctc cttgtaggca aggcttgtct 12660 tactttacct ttgtgacctc tgtgtctgttacagtgcctg gcactaagtc ggtaatttac 12720 tgaatgaacg atgggcccac tttgttgtgttcacctgtgc cgagctgagg ttcattgact 12780 cccactgcat ccaggggata aactctgacttagtcttgga gtggcagata caagcgacac 12840 acacccgtca gctggaaggc tcttgtcctctttgtgaatc agaaccctgt ttaggcttct 12900 aatcccacat caaagcccat ctcctccatgaaatactcag tttttctagc ctgcactgat 12960 ctcttccttc cctgaacttt caatgtatttaatgtacagc attgttcacg ctaccactgt 13020 attcgtcatt gattgtttca tatttgcaaaatgtttctct ccccagttca gttgtaggct 13080 ccttgtaatc cggcagccca tgccttatatgggctctatg cgcttacgtg ggcctctatg 13140 aaccctcaaa atgtcttttg ccaaataaaggccaagccag atggactgag ccctgtttct 13200 tctccttggc tgtggcctta ctatcttcctgcccctcgta aacagctttg cccctgggag 13260 tttgaggctg cagtgagtta tgatcgtgccactgccctga ggcctgggtg acagagcaag 13320 accttgtctc taaaaaaatt aaaataaaaaatttaaaaaa acaactttgc caccctaccc 13380 atcacacccc acccgcctga cctgcttcacaggctcccga cggccactgg cttccacctt 13440 ccccatcttt ctcttcccgc ctccatcttttaagctgcga accagcccct gttcctttca 13500 ctgtccagct gtcaatgagc ccagagtcccttagccacct agtgctttct tccccctcct 13560 gctgtctctt ggggtcttct ggtgtctgattctgtcagcg gggcaggtgg cagtacctgt 13620 tctagcattc aggccacttg gggctgatccacgcattgtt tatgttctag cctgcaggac 13680 agccaggtgg atggccctgc agcgtgggagccttgcccac ggagtgttga atttcttaat 13740 tgaaatgaat acacttaaat taaaccaaagttttgtagaa gaaatcctgg ccaggcacgg 13800 tggctcacgc ctgtaatccc aacactttgggaggccgagg cgggtggatc acctgatgtc 13860 aggagtttaa gaccagcctg gccaacgtggtgaaatcccg tctctactaa aaatacaaaa 13920 aattagccag gcattgtggt gggtgcttgtaatcccagct gcttgcgggg ctgaggcagg 13980 agaatcgcat gaacccggga ggcggaggttacaatgagct gagatcgttc cattgcactc 14040 cagtctgggc aacaagagcg aaactccgtctcaaaaaaaa aaagaaatcc tgttggcttt 14100 ctgtgcagtt ttttgatgcc attgtgacacagagaaactt tatttcagga actgcttata 14160 tcaacatcag aacttaagga aatgtctgagtactactttg atgggaaagg gaaagccttt 14220 cgaccaatta ttgtggcgct aatggcccgagcatgcaata ttcatcataa caactcccgg 14280 tgagctcttt ttttcattcc tttcttgtttttatatttgg caagtctttc ttcccggggt 14340 tacttactgt ttcatttccc atttaagaattagcatatac cggctgggca tggtggctca 14400 tgcctataat cccagcattc tgggagcctgaggcgggtgg atcgtgagat caggagtttg 14460 agaccagcct ggccaacatg gtgagaccccgtctctacta aaaatacaaa aaaaattagc 14520 cgggcgtggt ggcaggtgcc tgtaatcccagctacttggg aggctgaggc aggagaatcg 14580 tttgaatctg ggaggcggag gttgcagtgagccgagatca cgccattgca ttccatcctg 14640 ggaacagagc aagactccgt ctcaaaaaaaccaaacaaaa caaaaaacat gggggatttt 14700 taaaacaaaa ctttggaggg gattttaataaacgatagaa ggtatatctt aagaaacaga 14760 gcacgaggga gaaactacta cttactgatttttatatttt cctttattaa gcttttcagt 14820 tgtcccatta aatgttccct actagattacggggagttct taacttgagg ttcacgggag 14880 ttgtgggatg tctgtaaaca acttgacattgtatgcaaaa ttttgtgtat atttggattt 14940 tttggggaag gatttctatg gcttacagtttattctcaaa gggaagtagg gccctaaaag 15000 ctcaggaccc cgtgtttgtt ttctctctgaatcttgctaa ccatggtgtc tgggtagaga 15060 aagtaggaat ccacacaaca gtagactcagacttgacatg tccagaacac agagttgtgg 15120 tggatcctga gacttgcccc cagatcccccgtcagtgcac gactctgccc cagctgctgt 15180 atcaggtatg gttggtacct gttggccttcatttcttagc ctcttcaagg attgccttgg 15240 ctacaaagag tcctctcacc ttaggctgtgccccttcggg aggcagccca catccaggga 15300 ctgatagatg aagggccatt ctacctgcacaccctagagg gtgtttcagg ctgttgattc 15360 cagctcagct tctcctgcta cccagtcctgtatcctctcc ccctccctca ggtgtcagta 15420 atcccaaggg ataatcccga ataaacatcctgtcctttaa acttcatctc agagtctgcc 15480 tcctgcagaa cctaacttgc aacagagctcagcaaaaccc aagctcattt tattaaagaa 15540 cccagatcaa aaaagagctt tatgggctgggcacagtggc tcacgcctgt aatcccagca 15600 ctttgggagg ccaaggtggg tggatcacatgagttcagga gtttgaggct agcctggcca 15660 acatggtgaa accccatctc tacaaaaaatataaaaatta gccagtcgtg gtgacacaca 15720 cctataaatc ccagctactc aggaggctggggcaggagaa tcgcttgaac ccaggaggtg 15780 gaggctgcag tgagcagaga ttgcgccactgcactccagc ctaggggaga ctctgtctca 15840 aaaaaaaaaa aaaagaaaag aaaaagagctttatgataga tttctataaa attgcttcac 15900 tcactgaatg cagcacagtt atagtgtctgcatgtttctc agacaagcca aacctaccta 15960 gcctgctcag ctgcctcatt gaagacactgttatcactgg cacctggatc ttggcaccat 16020 ccattcctgt gtgaaactct tttagttctaagaagtgaat atcattgcca gcaatcagga 16080 taacagacta cccaaattgt gctgtacagagatctgttga tatcaatttt gcaaatagcc 16140 aatggcattg gattacatta gtccagttttcaaagctgaa ctagatgttt agggggtcaa 16200 attattagac acagttttca cagtagataacaggttgaga gtccaggtgt gataacaatc 16260 ctgtattcag aggaggtaac ttttatagcactataaaaaa ctaaggaaat taccaaagcc 16320 tatccctgaa aagactgtaa caaaacagccatttagcact gactggctcc agtgattcca 16380 aaggtcaggc cactaacaac tgtagagccattcctggttt ctgtgtgttc tgtgtcactg 16440 agccagccac tgctgctgct gtttatgggataggcaagaa ggttgctacc aaaggaaatg 16500 gttttgtctt tcacctagga gatggttctgtggatatgtc catagtcact gttaaaacgg 16560 atctttgaag tacattctac agtaagggacacccattaag ctggagattt tgatgagcag 16620 atggtcatca tttcatgcat gagtgcaaggtcaagcataa gaacctcagt gagagcaaga 16680 gggctgcagg gcttgtgacc gtggtgtgtgcaccttgttc agcaccctcc atgccaggat 16740 gaggctgatt ctgtctgcaa aggaatcaactcctatactt tttttttttt ttttgtcttg 16800 ctctgtcacc caggctggag tgcagtggcacaatctcagc tcatttcaac ctctgcctcc 16860 caggttcaag cgattcttct gcctcagcctcccaagtagc tgggattaca ggcacacacc 16920 atcacgccca gctaatgttt gtatttttagtagagacggg gtttcatcat gttggccagg 16980 ctagtcttaa actcctgacc tcaagtgatccgcctgtctc cccctcccaa agtgctgaga 17040 ttaaaggcat gagccaccgc gcctgggcactcctacactt tccttaccca tccccaattt 17100 gaaagaactg aatgctggtt agtccatagtaccctggacc ctatagagaa agctctgcac 17160 agagcaacct ggataagttg tagatccaggtatcatcata ggggtctctc tccacatatc 17220 ccaagattca gaagctcttg taatttcttcttcagtggca agcaaataag caatagtgtg 17280 aaccctgcat catttatgat gcaactttgcagacatccat tatatccaca gctaaatctg 17340 aaaatattca ggggttgctg cttttagataccaattattt tctcttgaca ttaaaatgct 17400 aacaaatcta tgactgttag gatcaagttaaaaaaattcc cactgaagag agacagactt 17460 tattctgagc cttacgtgtt tctttaggtttagaaggtga atgagcagtg gctggggagg 17520 atggcctaga agttggagct cacaggcttcctccctgcac tctgccattc cttagattgg 17580 aggcgccctt gatacagatc cctccctacacactgggggt ttacttgcaa tttaagactt 17640 cacattttat attagtatga acagggaaaatatattttgt aaaaccacat gtaaacctcg 17700 taaaggattc actggtaggg tcattatattattctgtcta tttttaggta tgtttgaaac 17760 tcttcattat taaaaaaatt tttttggccgggtgcggtgg ctcacgtaat ctcagcactc 17820 tgggaggcca aggcaggtgg atcatgtgaggtcatgagtt tgagaccagc cttgccaaca 17880 tggcaaaacc ccatctctac taaaaatacaaaaattatct gggcgtggtg gcacatgcct 17940 gtagtctcag ctacctggga ggctcaggcagaagaatcac taaaacaagg aggcgaaggt 18000 tgcagtgagt caagattgcg cccctgcactctagcctggg tgaaagagca agactccatc 18060 tcaaaaaaaa aaaagataat tttttaaatctaatgaagga ggaaagaaaa gtcctgacag 18120 gcatgctgaa tcatagcata ctcttgcaggtgtgaagtac agaggacgta gccaactctc 18180 aagaccaagg gcttcatttt ccatgctaccttgcctgtca cctctcccag atcctgggaa 18240 aatgtgatcc actatttcac agtaggaaatagaaatggtg cccagttttt tgaaggcttg 18300 attcagtttg gcattttgga gatgtcatcttaaggacagt gtgaggtttt tctgtaatct 18360 gtgcattttg gtcatctgtc ccaccctcatgttatggata agcagtggca gcatttccca 18420 gatgtaagct gacacacact aaagctgaactggataaaaa atacatcagg taaaactatg 18480 gaacatctga aatatgatgt atattctacgtagaagctgt gttacagtac caaataacat 18540 ttcagtttca tcctgatttc atcagtcaacaatttagcca tgcaaaatga cattttttat 18600 tctatttatt tatttattcg gagacagagtctcgctctac cccccaggct ggagtacagt 18660 ggtgcaagtc tcagctcact gcaacctccacctcccaggt tcaagcgatt ctcctgcctc 18720 agcctcccaa gtagctggga ttacaggcacccactaccat gcctggctag tttttgtatt 18780 tttagtggag acgtggtttc gtcatgttggccaggctggt ctcgaactcc tgacctcagg 18840 tgatcctcct gcctcggcgt cccaaagcgctgggattatg ggcatgagcc actgcgccag 18900 gcgcaaaatg acatttttag atggatatatagtctatgaa atttcaaaat attttaagaa 18960 atctttgttg taataatagc ttcagattaccaaaacaact ctagtatctt ggtgagtgct 19020 gccaatttca ttgcaacttc tcagcaggagccccgtctgc tgatgtaatt tatcataatg 19080 gaagtggtgc ccaacttctg aatgcatgagaaaggctaga ccttacctgt tgttttaagg 19140 taaggtctac tgctaactag taggaggtgtctaatttatt agactgaaat tcacttgcaa 19200 aaatattcta aaagccttat attaaaaaaaaactgtaaaa gtttatatct tttcctgtgc 19260 attcaactca aagaagatag ggcctagtaaatttacctga aaaatattta agtattctaa 19320 tataaaaact gaatctcact gagggattcaggtggcttaa aactcacctg aaccctgaac 19380 ctctattttc tcatttactg aagtttattggggtttttgt ttttttgtgt gtttttttga 19440 aatgaagtct ctgtcaccca ggctggagtgcagtggcata atctcggctc actgcaacct 19500 ccacttcccg gctcaagtga ttctcctgcatcagcctctc aagtagctgg gattacagat 19560 gcacaccacc atgcccgact aatctttgtattttaagtag agttggagtt tcaccctgtt 19620 gtccaggctg gtctcgaact cctgacctcaagtgatccgc ccgcctcagc cttccagagt 19680 gctgggatta caggcaggaa cctgtaactgtgcctagact acagaagtgg tttttatatg 19740 ctaatttgtc cctaccctcc actgcttttgttttaatact ccccccttag aagaatttgt 19800 tgtgatctag acatattaag aagttgtaactgaaatatta ataaagaatg aggccaggcg 19860 tggtggctca cacctgtaat cccagcactttgggaagctg aggtgggtgg atcacctgaa 19920 gtcaggaatt caagaccagc ctggtcaacatggtgaaacc ccatctctac taaaaataca 19980 aacattagct ggatgtggtg gtgtgcacatgtaatccaag ctacttggga ggctaaggca 20040 ggagaatggc ttgaacccgg gaggcagaggttgtagtgag ccgagatcac accattgcac 20100 tccagcctgg gcaataagag tgaaattccatctcaaaaaa aaaaaaaaaa aaaaggaatg 20160 agtagcactg tagacatgat ttccaggctgagagcagttg aaagggtcta gggtttagtt 20220 ctaaggctgc tggtaagagg agccagcgtgacatcatatt ttaaaattat atgtaaagca 20280 agatcaaaag ctttcctcat gctgatttagtgtcgatagt taaattacag caccttttat 20340 gtagttatac ttcatttttc attgctttctgccgggtctg aggaattgga atgagcatta 20400 ccttgtgcag atgttcagat tcgatttttaaagaaaaagt catatttcag aatccctctc 20460 ccttttttcc cctctaagat acaacctgatggtatttgaa aataagcatt tggaataagt 20520 gcaacatttg gttagtgtgt gtttaaatgaggatatgttt taggttccaa atggttattt 20580 cgccagtttg attttcttga aatttagtttttaaaaattg ccatagatga tggtggtaat 20640 aatgattaaa atgaaatggg ggacattccctctgaactgt aaaatttata tctgtgtcct 20700 gtcttcttga gcctacttat cctatagtttgtgttaaact tgggaaataa aagtttaaat 20760 ttctaatgag aaggttaaat gtgagttggaagaaagtttt acaaacatct tctgttggtt 20820 actgaggttg tcatactaaa cgtttaatttaagacattac tacgcggggt gcggtggctt 20880 acgcctgtaa tccacacttt gggaggccaaggcaggcgga tcacttgagg tcaggagttc 20940 gagaccaacc tagccaacat ggtgaaaccccatctctact aaaaatataa aatttagctg 21000 ggtgtggtgg tgtatgcctg tagtcccagctactcatgga ggctgaagtg ggagaatcac 21060 ttgaacctgg gaggcgaagg ttgtgagccaatattgcacc actgcactcc agcctgggtg 21120 acagagcgag accctgtctc aaaaacaagaaaagacagta ttaaaagcct tgaacattga 21180 gacagttgag tctttaaaat acttttaaaaaatgcttctc acctatcttc cctatccacc 21240 ccaaaattta attgtaaact tataaacttaaacacctgac caagaacact gttataaaga 21300 tgattcttca gcccaataag atcagccagacttctgatcg tttactgttt ttttggctaa 21360 tggtacaatt tctacttctt caatggggaattcataaaat gtagttgtgg cagggtttct 21420 catacatttg taaatgtata gaaatggctgtgtggtgaag ccagagtttt tataccgttt 21480 ctcttagaga aataacattc tttatcctagatccgatgtc cagttttcac aagctgattg 21540 ctgagaaggt tctaggcggc gtctgttaaaaagcattgct ttctgttaat tagacatgtg 21600 caagccagcc agcgcgccat agccttaattgcagaaatga tccacactgc tagtctggtt 21660 cacgatgacg ttattgacga tgcaagttctcgaagaggaa aacacacagt taataagatc 21720 tggggtgaaa agaaggtatg gttttttggtttttttaaaa tctctcttac tgaatcacac 21780 gcttttcgga ccgcatttgt ttctcagatttgtctcatta aaaatatgct tgctcaaatg 21840 taatgtggtc ttctgaattt caaaaaagtattcatgtctt gtccaaatac agatatttga 21900 taaataaata ataaaaaata ccatggaaaaataaacttag tatttctaat agaattcctt 21960 tggttataag gaaagggatt ttcatgggtgtccaaaaaat gtatttcatg aggaatcata 22020 cgttttactt ttgggcttag attacccagattcagtttaa ttttttaaac atttgtataa 22080 ttgagtgctg catataaatg ccaaagcaaacaaataaaac taataaaaga aaaaagaaac 22140 ctcagagaga tactgttctg ccatgaaaattctgtctttt gaaatagaag ttctgtaatt 22200 gggtttggtt catatatgta tatattaaagcatatttcta tattattagc attgggaata 22260 tgggaaacag ggacttggtt tgaggatgcatagatcctgg gttgaaggat gagaataaag 22320 ttgaacagat gagaatgaaa atgcacaggcatccatcgcc atcaccacac acgtgctcta 22380 caaacaaaaa gtttgtgcag gccaggcgcagtggctcatg cctgtaatcc cagcactttg 22440 ggaggctgag gtgggtggat cacctgaggtcaggagttcg agaccagcct ggccaacaag 22500 gcaaaaccct gtcactacta aaaaaacacaaaaattagcc agtgtacacc tgtaatctca 22560 gctactcagg aggcttaggc aggagaatcgcttgaacctg ggagagggag gttgcagtga 22620 gccaatatca cactactgca ctccagcctgggcaacagag tgagactcca tttcaaaaaa 22680 aaaaaaaaag tttgtgcaaa caactaccccaccctcacct ccttttctct catagattta 22740 tagtattcct ggttcattcc tatttaattctccttattaa aagaagagat atatgtatat 22800 acacacacac acacacacac acacacacacacacatacat atatatatgt ttattgttat 22860 cttcacatgc tggttttatt tgaacataaatgactgtttt gaaatcgagt cttgcaattt 22920 catttatatg ccttttattt caaattttagattaaaaggt tttacgtcct gttgactaaa 22980 ttctgtacat cagaatgttg gccaaaaagcagatggcgtt tagatttgga gaggatgtgg 23040 gtaactctat gacccatccc tgccgtcagctgtatctgtt ttcaggcata gccagtccta 23100 aagccttatg tggagccttg ggcgggggaagaggatcaag agaacaaatg atggtctccg 23160 ccttggctag cccctgtgtg tctgcctctgccactgggga cctctttctg agggcaggtc 23220 aaggacacat gtgccgcctt cacctgcctctttcagttct gacagccatc tgcttagcac 23280 agggctactt gccagttcct acctgtttctgcctctgacc cacaactgct atgttaccgg 23340 tacaaaaccc cccacacaca gtgccccctcggtgagccct tgttaggcct ggcctgggct 23400 tcctagcact tctttccttt aactcccacccctggccgtc acagtcctgt ggcttccatg 23460 tcatatgctg gacctttggt ctctaggatctccccggcca ggtgaagaag gaactaaagg 23520 ccaaaggatc ccgagccttg agctgctaatgatgtagggg ctgggtcagg gaatgtaagt 23580 gggtacctat atatcataat ttgtaaaatgactttatagg catatactta catcaggatg 23640 tcttacataa tatgtatatt atataaagtggtgattaatt ggtaaacaca atgaacatca 23700 ctgtttagat actgaagaac ctaagacaataagtacctaa atagttatgt tgaaaattct 23760 gtgaactcta gcctttataa ctaattactacagaatgtaa cacttacggc cgggtgtggt 23820 ggctcacgcc tgtaatccta gcactttgggaggccgaggc agttggatca cttggggcca 23880 ggagttcgag accagcctgg ccaacatggtgaaaccccat ctctagtaaa aatacaaaaa 23940 tacctgggcg tggtggtgta cgcttgtagtcccagctact catgaggctg aggtgggggg 24000 attgcttgaa cccaggagga agagtctgcagtgagccgag gtcatgccac tgtactcaag 24060 cctgggcaac agagcaagac tctttctcaaaaaaaaaaaa aagaatgtaa tatatatctt 24120 taaatatgca aattaatgtg atatatagggtgtatttggt tatagcatat aattcaacta 24180 tttatgcagt agtttataac tagttgctaatacagtgtag acatccatca cagtctaaag 24240 aagactggaa atatgttgat tgcttagtgttcaatggaaa aacttttttt tttttttgtc 24300 taggaatgag gggcagtgcc acatagtacaaaaagcattt cacttggagc caaaattctt 24360 tggtttaatt tttattgaaa ataacatgtatcgtgccatg ctctggggtg tgctcagtac 24420 tagctgagat gattcctagt ctgagctcccactccactcc accctcccca ctgccctggg 24480 agcatagaga ctagagcatc actgactggtacgcaaggtg tgatggtcgc agcatgcatg 24540 ggtgttgtac acgcacaaaa gggaggcatctgaccagact gagggatcgg ggggaacttc 24600 ttagaggtgg catctgaacc aattatgcaaataagtccac tcagtgaaga agggggcaga 24660 actttttgag cagaaggaac agtaagtggaaaggcacaaa ggaagaatga acatccagtg 24720 tagtgaaact gaaggcagct gcattttggtggggctggtg agtgaagctg ctctccctct 24780 ccaggcattg atttcttcag gtgtcaactggggagatgga catggtcatc tctgagagcc 24840 aatacagttc aagtactgtc ccaaatctgtaaccgattat tagaaactgg gaagagtgac 24900 atggtcatct ctgagagcca gtccagttcaaacactgtcc caaatctgta actgattatt 24960 acgaactggg aagtgacatg gtcatctctgagagccagtc cagttcaaac actgttccaa 25020 atctgtaact gattgttacg gactcctgttgaggaagaag aaggtaaaaa accctgcatc 25080 caagatgagc ccccacttcc acgaagctcccttccagtca gtttcatcat tggctctact 25140 gctctgatgg atttttcaga acgttctgttttccccctgt ctttttctag gctgttcttg 25200 ctggagattt aattctttct gcagcatctatagctctggc acgaattgga aatacaactg 25260 ttatatctat tttaacccaa gttattgaagatttggtgcg tggtacgttg attctgattt 25320 ttcttctttg ttattcaacc ctggtgtttagccaggcaat aaagccacct ctcaaatgac 25380 tcctttcctt ctttataggt gaatttcttcagctcgggtc aaaagaaaat gagaatgaaa 25440 gatttgcaca ctaccttgag aagacattcaagaagaccgc cagcctgata gccaacagtt 25500 gtaaagcagt atgtacgttc tgtctttcttcaagttaaag cctcatagct cttttttggg 25560 agctaatttt cctagaaaat atttcggtgaagaatcttaa aatagtaccc aaaaatccca 25620 agaggttaat gaggaaaaga atgacatccccaaacaatag aggacttctg ctgtgttttc 25680 atttttgcca tcttcttttg gtatgcaggcgttgactttt catcttttct tcccaagaat 25740 tcaatcaaaa taagctttcc cgcaccttccccaatctgat tgccaaactg tatcattttg 25800 aacaatttat cataatttct ctaatgattgatatcacaca cactctttct gacacttcac 25860 cttttagaaa tgaagtgctc tgttctttaatataatattt actcaggaaa agatatctgt 25920 tagattgtac tagcattcta tgaacttttttttgtttttt tgttttttgt gtttttttga 25980 gacagagtct tttgctgtgt ggcccaggctacaaagcagt ggtgtgatct tggctcacta 26040 caacctccac ctcccaggtt caggcaattctcctgcctca gcttccctag tagctgggac 26100 cacaggtgtg cgccatcaca cccagctaatttttgtattt ttcataggga tgggggtttg 26160 ccatgttggc tgggctggtc tcaaactcctgccctcaagt gatccaccca ccttggccct 26220 gcaaagtact gggattacag gcatgagccattgcacctgg ccaatacatt gcacctggcc 26280 aatccttcta atatttttac atgaaatataaacaagtcct atttcttcag agtacaaatt 26340 gagtataagc cataactgtt tttccccttgctttctccct ccctccgctg tgcatacaca 26400 tgtatacatt ttttttttaa tgagtaatacctttaatccg tagacaaatg tgtggtattc 26460 atcgttagtc caagaatgaa aagcagtctctccatagaat tgtttatctg cccatctcta 26520 agcctgacag atacacagag acaaaacctggacaaatgac attcccatgt aattacagcc 26580 acaaaataag gaagacctgt aagggtcgcatgtcaattgc tgtcatgata gactcctaac 26640 ataattaaca ggtaaagaga gcttttgctcagacctctca gatgaaaaag tctcttgctg 26700 ttagtgtctc tgttttgaaa agtgtcaagaaatgtataat tgcaaggcag aaaagaatga 26760 ggacaatctt ttcttcctaa aaagacctaatagaaacttt aaggaatgtg aattatgtag 26820 aacatgctag ccacagtctc gaccacttttgtctttttat taaaagggct attatgtttt 26880 tatttccaag aaattatgtg ggttttttttttaaagtgag atggaagaaa gtatagacac 26940 gagatgctaa ataagagaat agcctattttaagtgggtgc tctaacactt ttacagtaat 27000 cctttacata ttatcatgcc cttgatggcccaactctccc tgaaggtcag gatccatcct 27060 cttaacacat tagggtgcct taatattacttactaatatt tatccttaag aggatgtgtt 27120 aagtgaggct cattgataat ttcacaatttgagactgcaa acttagaagc attagcatgg 27180 tcaggcaagg tggctcacac ctgtaatcccagcatttagg gagactgagg cggaaggttt 27240 gcttgtgacc agaagttcaa gaccaacctaggcaacatag caagatcccc atctctactt 27300 aaaaataatg tttaaaaaat aaagtcttagaagcattagt agtagtgaga ctattggaat 27360 tggaaacacc aagacttact gtctgcaccatgcagacatc ctgcaggcac ggggtggggc 27420 ggcaccaaat tggagctagc acagaaattcactcagtgat ggaagcttac aaagggtcca 27480 aagaaatgga cctgagtcat gatagagaggtttccttgtg gtcactgttt tctgtttaag 27540 aagccaaaga taatgcacag gaattcttttataaagatat gcacctcatt cttcaaagat 27600 tagcagctga acgaacagtg aacatgttaacgtggctgga cccttaataa aaatgaaatg 27660 tttcatgctg cccactaggg ggcatgctggcatcgtccca gcactacctc cttttcatgt 27720 ggtttattcc taaactccac agctcttagaataataaagc aaaatgatag tgtgagctat 27780 ttgaataaaa gtttctatat ttaagtgcctatgggtggaa atattccaga ggtgttatgg 27840 attcaaaatg gctattttta cgtactcttggtatttaaaa tgcaaagcca tgcgagctcc 27900 aaataaatgc atgcaaagca aattagacacaccaaaaaga ggggaggagg agaactgaaa 27960 gagcagaatt attacagaag aagaactaatgggattgcaa aatgtattga gaattggagg 28020 gaaacttaca agctgcattc tactaaggataccatttctt catctccctt ccttttttta 28080 gtgaaaataa tattaaaatc taagagagggctgtctaggg ggattgtttt gtgattgact 28140 gatattaaaa tagaatccat tttaggccgggcacggtggc tcacacctgt aatcctaaca 28200 ctttaggagg ccgaggtgag tggatcactttagtccagga tttcaagacc agcctggcca 28260 acatggcaaa accccgtctg tactaaaaatacaaaaatta gccgggcgtg gtggtgcacg 28320 cctgtaatgc cagctacttg ggaagctgaggcaggagaac cgcttgaacc cgggaggtgg 28380 aggttgcagt gagccaagat cactccactgcattccagcc tgggcaatag agcgagagtc 28440 tctcaaaaaa aaaaaaacaa accagaattcatgttattat caaaagatga cttatttatt 28500 tacatactta ctcacttgca aaatacattttgtactcatg caaaatacat tttgtagctt 28560 actaataaag acagtggctt gtttcccaggaaatctggtg gaaatgagac ctgagaggtc 28620 agagggcctg tccagttgtt gttaagccagacagtagctg agttgagact tgaacccaga 28680 gctgggtatg gtaatcctgc cttgtttctctctctctctc tctctttttt tttttttttc 28740 tgaatttcta ttttctccag ggctgcttgtggcctggaat taatgggctc tcttcctatt 28800 acttgatttt caaagcctca gagtaccactacagaattgc atattgtggg tcacattagc 28860 agaacactct tttttttttt ttttaattcatttttttgag atggagtctc gctctgttgc 28920 ccaggctgga gcgcagtggc atgatctcagctcactgcaa cctccacctc ccaggttcaa 28980 gtgattctcc cacctcagcc tcccgagtagctgggattac aggtgcacac caccacatgc 29040 cacacctggc ctcttttttt tttacaatattcatatctat ctataggcct cattcagatc 29100 ttgccagttg tgaactgtta taacgaaagggaaaacatat ttttctgttc cagcatccag 29160 tccaggattt cacgttgcat tttgctgtcatgactctgta gtctcttgtc atctagaaca 29220 gtctttcttt gcctctcatt accttggtatttggaagagt gcaggccagt tatgttgttg 29280 agcctctcag tcgggcttct ctgatacttctcaagattcg atccaggtta tgaatatttg 29340 gcaggaatac cacaagagcg gtgctgtcctcagctcctca taccaggagg cgcgtgctgt 29400 cttgtctgtc ccgttactgg tgatgcatgcctggatcgct tgattaggat actgtcgggc 29460 cgggcacagt ggctcacgcc tgtaatcccagcactttgga aggccgaggt gggcagatta 29520 cctgaggtca agagttcaag accagcctggccaacatggt aaaaccccat ctctactaaa 29580 attataaaaa attagctggg catggtggcgggcacctgta atctcagcac tttgggaggc 29640 cgaggcgggt agatcacctg aggtcaagagtttgacaagc gtggccaaca tagtgaaacc 29700 atgtctctac taaaaataca aaaattagccgggcgtagtg gcaggcgcct ataatcccag 29760 ctactctgga ggctgataca ggagaattgcttgaatccgg gaggcggagg ttgcagtgag 29820 ccgagattgc accattgcac tcaagcctgggcaacaagag tgaaactcca tctcaaaaaa 29880 aaaagatact gtctgccagg tttcttcaatctaaaattac tattttaacc tattttgggc 29940 aaagtatttt cagattatgt aaatattattctgatggttg tcaaatggct gtttttctat 30000 tttcatcatt tcttctatat cactcttctacaagaaagtt ggtatcctat tttttttgtt 30060 attattattt cattctcaaa agagctgaagagttggtatt ccattgtaag gaagctctta 30120 actgtatggg ctcttggatt cttattttattctgtatctt tttttttttt tttttttttt 30180 gagccagggt atcactctgt cacccacgctggagtgcagt ggtgcaatct cagctcactg 30240 caacctccac ctcccgggct caggtgatcctcccacctca gcctcctgag tagctgggat 30300 tacatgcggc tgccaccata cccggatgacttttttgtat ttttagtaga gacggggttt 30360 tgccattttg cccaggctgg tctcaaactcctggcctcaa gtgatcgccc acctcggcct 30420 cccaaagtgc tgggattata ggcgtgagtcaccgtgccca gccatcttat tttattctgt 30480 agattataac actgtcatta ttttaatactcaattatcca acgtatggcc agtaggggag 30540 gccctttaac ctggctctgt gtcctccatcattttttcag caccccattt ctggcaggag 30600 atattcagcc ctgttattca ctgttctccaaggagcccac attccttttg gtggaaaatg 30660 gtgttaagaa accagaattt gagtactgggtgtcatcgtt tctagaccca ctcaatggac 30720 agagctagga aatacatgta tgtattatatgtaatgtagg tagatacgtg tatatctttc 30780 tgtacccatc catacttatt caaaaccacgaggtatcaaa cagatagttc caaatccatc 30840 taacaccaca ggatttgtgg tttattccaggattccatct ttccatattt gtaaccccct 30900 tcccaaacag tgagagacct ggctcctcttctccgcggtg tatttatttg cacaatctga 30960 gaatatacca taggagttta caaattcatgactcatatct ctgtgaaaaa caaacccagt 31020 agctagaatt cagtatttat cattccttttgtcttgggcc tgaggataat agaatcaaag 31080 cactgttcaa aagttacctt tctctacgtatcagtgtggt tgtgttatta tttggaatat 31140 attaacccat ttatgcctag tgttccattattggaatgct aagcttgtgg agttatttct 31200 atcctactgc tcaaggtcat taccaaggtctgatttttca caaaacaaat ttgcaacctc 31260 cagcataaat gggttaatag ttggttcctttttttttttt tttttttttt tgagacggag 31320 tcttgctctt tcgcccaggc cggagtgcagtggcactatc tcagctcact gcaagctctg 31380 cctcccgggt tcacgccatt ctcctgcctcagcctcccga gtagctggga ctacaggtgc 31440 ccgctaccac gcccggctaa ttttttgtatttttagtaga gatggggttt catggtgtta 31500 gccaggatgg tctcgatctc ctgacctcgtgatccgcccg cctcggcctc ccaaagtgct 31560 gggattacag gtgtgagcca ccgcgcccagcctagttgga ttcattttta ttccacttta 31620 gggattccct ctcattcttg ctgatttcgtgttttggttt tgagcgtttt gttattgttg 31680 ctgttgtttt gaatgtttga aacaacgtggctgggcacag tgtctcatgc ctgtaatcct 31740 agcactttgg gaggccaagg cagatggattgcttgagccc aggagtttga gaccagcctg 31800 ggcaacatgg cgaaacctct gtctacaaggaatacaaaaa ttagctgggt gtggtagtgc 31860 acacctgaag tcccagctac ctgggaggctgaggtgggag gatcacctga gccctgggag 31920 gtcgaggctg cagtgagctg tgatcacaccactgcactcc agcctggcaa cagagtgaga 31980 ctctgtctaa aaaaataata ataataaaaaaaataaaaga cattaatgta gctccaaaag 32040 tcagaactat acaatacagt aactctcccttttgccctgt tttcttcccc tacccccttg 32100 taggtaagca gtctcgttca cttctggttcctccttcctg ggtttcgttt agcacaaaca 32160 ggcagacaca agtatgctat cttcccttctttctcacagc aagaagtagt accctagact 32220 actctgcttt ccttttgcat ttcttcagttaacaatttat taggaaaatg attccacatt 32280 ggtttgtagg atcttcctca tttttaaaaccactgtatca gggctgggct tgtaatccca 32340 gcactttggg aggctgaggc ggccgaatcacgaggtcagg agttcgagac cagcctggcc 32400 aacatggtga aaccccttct ccactaaaaatacaaaaatt agctgggcgt ggtggcacac 32460 acctgtaatc ccagcacttt gggaggccaaagcaggcgga tcacttgaga ccagagttca 32520 aggccaggcc ggccaacatg gtggaaccccatctctacta aaagtacaaa aattagccag 32580 gtatggtggt gggtgcctgt aatcccagctactcgggagg ctgaggcaca agaatagctt 32640 gaacccggga ggtggaggtt gtagtgagccaagatcacgt cactgcactc cagcatgggc 32700 gacaaagtga gactccatct caaaaaaattaataaataaa taatttaaaa atttttttaa 32760 aagcctctgt atcagatccc attatgtggacatagcatgg tttattcagc caatcttcta 32820 tacatgggca tttaggtttc cagttttacaattacaaaca ctactgcaag ggctgatcgt 32880 gcataaacct atttttgtgt tgtgagaggagtgcctgcag ggtgaattta ggggtaattg 32940 cacatgtggt tttgttaggc accaccagatttccctctgt tgggtctgta ccagtatgtg 33000 tttccaccag cagcttttca gaacctcttttcccacagcc ttactaacta aatgtgccat 33060 tacacgcttg agtttttgcc agtccagtagaggagaatgt attgcagtgg agttttaatt 33120 tacatctctc ctttctgagt gaggtggaacatttttcaga tgcttaaggg ccatttattt 33180 atttgtttat ttatcggagt ctcactctgtcacccaggct ggagtgcagt ggcacgatct 33240 cggctcactg caacctccgc ctcccaggttcaagcaattc tcctgcctca gcctcccaag 33300 tagctgggat tacaggcgtg agccactgcgcccagccctt cccagtctcg ttgagacgtg 33360 gcttgtaagg atgtgatgca aggtgtgaaccttccccaga aagaagtgca tacgcacagc 33420 acagcctggc tgcagcatct gcttcaaacaaaacagttag ccgagctcca ctttctttga 33480 cttaatcatt cttcatgctc cacccatttccggaaagatg tgaggcatcc ctctgatgct 33540 gagcgcaagt ctgtgcacct ctttgccttcttgtatgttg tgctatgttg cgtccaattt 33600 gtggcagttg ttttctgttt tcctgctacagatctaggca gtactgacct gggatgggga 33660 agcaggactc tctggcatgg tctttatgctaccccaggga aatctgggat agttgaaatc 33720 tgagcagtaa tgtcccttcg ccatgtgtcaggcagaagct gcccactaaa taagctctgt 33780 tcagcatgtg tgggcacaga ggggacagtgcagacagact gcagctttat gcttacttgg 33840 acacattcct tgacctcttg gaggttctattttgttgccc agcatagaag cagcactcct 33900 gtgttgaagt gatgaagaga acagtgtctggagccagacc atgtggatga aattcaggtt 33960 ccagcgtgtt ggagccatgt aatgcctggtaccgtctgtg tctcagtttc tcgaacttca 34020 taagtgactt aagatgtgag aactctcagaacagtgccta gcacatagtg gacgctcaga 34080 aattgttagc ttttatcatc accaaccacataatccatat ttgtgggata ttgtgaggtt 34140 taactcaaat catacaaagt gttcatcatatgtgctccac acattttaat ccattaaaat 34200 gttcactaac agatttttag gccgggcacggtggctcacg cctgtaatcc cagcactttg 34260 ggaggctgag gcaggcggat cacctgaggtcaggagttcg agaccagcct gaaaccccgt 34320 ctctactaaa aatacaaaaa ttagctgggcgtggtggcat acggaggttg aggcaggaga 34380 atcgcttgaa cttgggaggt ggagattacagtgagcccag atcgtgccgc tgcactctag 34440 cctgggtgac agagagactc cgtctccaaaaaaagaaata gatttttaga aaatctttca 34500 cttttcagga agaaagctta tagtctctgtggctctgtgt tgaggaaaca gcattaatct 34560 cactacagga gactgcttca ctatcagttatttctacgtg gaaatatctt gattcccagt 34620 atcattctgt cctgagccca gcacatcccaggctgcccaa atcttgccat gctctccttt 34680 gagccaagct gatcttagct tctctcgaaagtttctgaat tgtcccccat atggtctccc 34740 agactcttca gttgaaaaaa ggagccctccctgacagccc aggggtcggt gcctgctcat 34800 gggaaggtgg ttgctgttga aagcagttatgagcttactg ttcactcaac tcagtggcca 34860 cctgaccctt tatggtgcat gcaattttaccatgtactta tggccaagca ctacatagtc 34920 aacagacctc attaagttgt caaaaagcattctcaggctg aggacgttag gcaacctggc 34980 tttagttggc agaggtgcgt ggacactgccaaggctccta tttctggttc cagtggatga 35040 ggtggaggag gattatttgt aataatagcaaacagccagg tgcggtggct cacacctgat 35100 aatctcaacg ctttgggagg tggaggtgggaggatcacga gcccaggagt ttgaggccag 35160 cctagacaac atggtgagac tccatctctatgaaaaaatt aaaaatcagc tgggtgtggt 35220 tgcgcgtgcc tgtagtccca gctactcaggagactgaggc ggaaggaacc cttgagccca 35280 ggagttcaag gttacagtga gctatgatcgcaccactgta ccccagcctg ggcaacagag 35340 tgagacccta tttctaaaaa gagataataatagcaaacac acattgagtt ctaaccaggt 35400 gccaggcagt atactgaggg cttaaatgcagcatcatgtc tgtttctcac agcaacccta 35460 caaggtaagt gcttgtgatt tctacattgtacagatgagc aagagagatt cagtaacatg 35520 ccgaggtctt gtagagggca caaatgcagccccacagtct cacagcagag cccgcagcac 35580 tgcaccacac tgacgcctga gcaaagttcactccctgact ggagagccac agaggcacga 35640 ccgaaggtca ggggacaggg tttcctagcatccgcgagcc ttacagaaag gcaactgtgc 35700 agtgctccag ctggctttct catggagagtcaacagagac atttcccctc cagtagaaca 35760 cagaccgtct ctcccctccc ccttgttggttttacccagg ctttgttttc tgaaaatgtg 35820 gctgggcctg cttaacatgc ttagcagggcactgggaaat gcacttcagt ggccggtgcc 35880 agctagcttt ttggagtttt aaaaagactttcagaagtct tatttctccc ccattgaaag 35940 gagggaaaag ggtttttata cagttacttcttttgagaga aatgtggaaa cagtgggacc 36000 agtgaagttc cttccgataa tgaaagagcgatatctgtgt ctgaagcagg aggcttgaga 36060 tgatttttat ggacacacca agaaataactgcattcagaa acaggtgaaa ttcccaacga 36120 tgatgaaaag aaaggactac agatgggaaaattgtgtgtg attacattag tatctcttcc 36180 tgaaatgagg gatacattga tagagatgattaaagccaac agtaatcggg ctagcttgcc 36240 gagtgctaga agtcagtatt tcacagatgggggtccgttt cttttgcatg tcaagaaggt 36300 ttacttagca tgttaccagc agaactagtccagttgtagc tcagtttttc ctaagcagtg 36360 ggaaaggctg cttatcctgt ctgaaagcaggggttggaga aggagaattt tcttagaatt 36420 taacaacaca atctgagact gaaattcttgactggaaatg cggttttgta catgcttggt 36480 gtccctctga tgtcagcatc tcctgagtgtgtataatcta gccccgctgc ctcctatttt 36540 aaggaattcc ttcagccagg ggtcagctgctttgttgctg cctggaagca gcttatctca 36600 gaatgctctt tctgtttcag gtctctgttctaggatgtcc cgacccagtg gtgcatgaga 36660 tcgcctatca gtacggaaaa aatgtaggaatagcttttca ggttagtatg ctttttattt 36720 gtaagaatgg tggcgtagtg atacagtcagcattctcccc tagtgtgtaa tcgtcaaaat 36780 agtaagaacg atggcagcag tgttggcatggcggtgctcc ttacatccca tttttccttt 36840 tgccagctaa tagatgatgt attggacttcacctcgtgtt ctgaccagat gggcaaacca 36900 acatcagctg atctgaagct cgggttagccactggtcctg tcctgtttgc ctgtcagcag 36960 gtaggtttta caaactccct ttgacacatcactgcatagc cccacagaac tgatgtcccg 37020 cggcacagct gatgggaaga ttgcataaaggaatagatgg gaaggcattc agataagaga 37080 tcacaggtct gcatttgatc ctggctgagtgagatgttgg ggctggtcat ttcaccttgc 37140 taagactgtt tccttatctg taaaattgagaagatcacct ttctcccagg gtggttgtga 37200 ggattagcta agatcctatt tgagatctttgtgtcttgtg gtgtgccata ggcatttgag 37260 gtagcatcgt gattatttcc atatattttggccactggca aagtgaacgg tttctaagtc 37320 ttgattatag gactggactt tggtggtcctcagagcccct taaaaggcat aggaagcatc 37380 aagggcctcc aagcataaga aattctccggttctagaagt ttaatgagac tctgctgctc 37440 tgagagaggc tttagaacct cggccattgcctcaaaatgt caggaagtca gtggagtgca 37500 gtagacccac atagttcctt ctttctccggattgagggac tgagtccccc ttaatgtgaa 37560 tgaaaggctt aggaagcttc aaagatgttccctcgactga caaagcagac attctcacag 37620 cctcctccag accctgccac atggcttgtggctgtactga atgttacttg aaataagtga 37680 gacattagct ggtgttggaa catctcgttaatagattttc atcttagtag tatttaattt 37740 gttatgttgc aaagcagtaa gatgttcatcaccgtgccat gaaattcaac attagctctt 37800 tggtgtaaaa ttatagtaac ttttggtctttcagagattt tgcctctatt ctgtcttcac 37860 gtttacaaag gtcagtcatg tcctccataaaattcagtga ttccactgtg atacagaaac 37920 cacggccctt gcttttggtg ggtttctgattggagagagg aaaggtcatc tttcacccac 37980 tatctagcat agccattggc agcatgattcttcccagggg aggctgacgt tctgggtggc 38040 tggaccaggc tactttggca gcttgctaaggctatgaatg gagatgttgg ggtactcggt 38100 aggaacaccc gccctcatta ttacaaggcttccatcctct caaactttgg aggctgaggt 38160 aagaagtgaa aggtatgctg taaataggtcctctctccca atgaggctta cttgccagcc 38220 caaaatcaaa gagtataata catgtgcccagttttgacaa aaatttataa aacctccttt 38280 tgtacattaa ggcaagagtg aggaacatttgagccatgta ggtgttatgc tggggattag 38340 aaaaatgagg cactggctac cagtaacctatataactgcg aacattactt ctcagatact 38400 tgttagtaaa catgagtgaa ggaaagcaagatggactgag tgtgctgaaa tccagctagc 38460 ttggtaaaga ttcctttacc taggctcagattatcaggat aaaaggaaaa agcctttttc 38520 cctggagaag tctatgagaa agttttggttgctctatttg taaaaatctt caaattgtta 38580 agtacttgtt atgaacccca ggatactaagttaccggttg agtcctactt aaaccttaag 38640 gtgactgggt gagaggaggc tggcctcttcggactgtgtt tcactctgaa tatatttcag 38700 aagaaactaa cttactttcc cctacacacacaaaggagta atggctatct ctgctttcat 38760 atatagtggg ggaaagggga aatggacctctgcatagtat ctgtcagtaa tctacaagag 38820 actgaaaaat gctggttagg cggtggctcatgcctgtaat cccagcactt tgggaggctg 38880 aggcagttgg attatgaggt caggagttcaagaccagcct gaccaatatg gtggaaaccc 38940 cgtctctact aaaaatacaa aaattagccgggcctggtgg tgcatgcctg taatcccagc 39000 tactcgggag gccaaggaag gagaatccttgaacctggga ggcagaggtt gcagtgagcc 39060 gagactgcac tccagcctgg gtgacagagtgagactccgt ctcaaaaaaa aaaaaaaagg 39120 ctggttaaaa aaagcaagca aaaggaaaaaaaaagattac tgtacccgaa gccatggttt 39180 tatgtgtgct ttgctgggaa atcccagtcatgaggcacct actcatgctc accagacagc 39240 agtgttctca tctgcccata aggcagtgagttgaaaaggc acattgcagc ctcagaaaag 39300 ggaacacaga atggagtcca agcaggaagtgactctggac aggactcatt tcaaaagtag 39360 actgatgttt ctgtcttgtg gcacatgggccagaagttag ccaagtatgt atttataagt 39420 tgccttctaa taaaacagca aggttaggctcttttgtgga atacactgta aaacaagaga 39480 ttcttagcaa gaaatgtgtc aaaagatatatgggactaag attaattcag gtaaaaacaa 39540 gttccaaaaa taacgtaaga atatgcaatatctccactta atgaaaatgt gtttttagtt 39600 tacaaaggat tctttcatac attatctctaatctcacaac tcctctttgt ggtagatatt 39660 atggtgttta ttctgcaagt aagaaactgatgcccaaacc tcgccaaaat taaacatctg 39720 gtaaatggca cagcagggaa ctgaacaagtctaagaccag tattcatttt attacatcat 39780 acatagtgtt attgccactg gtaatgctgagaagttagta gctatgatac cacacaggcc 39840 ttcccacaga gcaggtaact aacccacctgggcactgacg atactcaaag aatcatctct 39900 gtgtcatgtc tttgctattg taaacaacatacctacttgg tggagtagtt ctaagacgtc 39960 tgtaatcctt tccctttggt ag 39982 4308 PRT Homo sapiens 4 Ala Gln Ala His Arg Gln Lys Gly Leu Asp Leu SerGln Ile Pro Tyr 1 5 10 15 Phe Asn Leu Val Lys His Leu Thr Pro Ala CysPro Asn Val Tyr Ser 20 25 30 Ile Ser Gln Phe His His Thr Thr Pro Tyr SerLys Thr His Ser Gly 35 40 45 Glu Lys Tyr Thr Asp Pro Phe Lys Leu Gly TrpArg Asp Leu Lys Gly 50 55 60 Leu Tyr Glu Gly Ile Arg Lys Glu Pro Leu IleSer Thr Thr Glu Leu 65 70 75 80 Lys Glu Ile Ser Glu Tyr Tyr Phe Asp ValLys Gly Lys Ala Phe Arg 85 90 95 Pro Ile Ile Val Val Leu Met Ala Arg AlaCys Asn Ile His His Asn 100 105 110 Asn Ser Arg His Val Gln Ala Ser GlnArg Ala Ile Ala Leu Ile Ala 115 120 125 Glu Met Ile His Thr Ala Ser LeuVal His Asp Asp Val Ile Asp Asp 130 135 140 Ala Ser Ser Arg Arg Gly LysHis Thr Val Asn Lys Ile Trp Gly Glu 145 150 155 160 Lys Lys Ala Val LeuAla Gly Asp Leu Ile Leu Ser Ala Ala Ser Ile 165 170 175 Ala Leu Ala ArgIle Gly Asn Thr Thr Val Ile Ser Ile Leu Thr Gln 180 185 190 Val Ile GluAsp Leu Val Arg Gly Glu Phe Leu Gln Leu Gly Ser Lys 195 200 205 Glu AsnGlu Asn Glu Arg Phe Ala His Tyr Leu Glu Lys Thr Phe Lys 210 215 220 LysThr Ala Ser Leu Ile Ala Asn Ser Cys Lys Ala Val Ser Val Leu 225 230 235240 Gly Cys Pro Asp Pro Val Val His Glu Ile Ala Tyr Gln Tyr Gly Lys 245250 255 Asn Val Gly Ile Ala Phe Gln Leu Ile Asp Asp Val Leu Asp Phe Thr260 265 270 Ser Cys Ser Asp Gln Met Gly Lys Pro Thr Ser Ala Asp Leu LysLeu 275 280 285 Gly Leu Ala Thr Gly Pro Val Leu Phe Ala Cys Gln Gln PhePro Glu 290 295 300 Met Asn Ala Met 305

That which is claimed is:
 1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids.
 2. An isolated peptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids.
 3. An isolated antibody that selectively binds to a peptide of claim
 2. 4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).
 5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acid molecule of claim
 5. 7. A transgenic non-human animal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising a nucleic acid molecule of claim
 5. 9. A host cell containing the vector of claim
 8. 10. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.
 11. A method for producing any of the peptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.
 12. A method for detecting the presence of any of the peptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide.
 13. A method for detecting the presence of a nucleic acid molecule of claim 5 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample.
 14. A method for identifying a modulator of a peptide of claim 2, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide.
 15. The method of claim 14, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide.
 16. A method for identifying an agent that binds to any of the peptides of claim 2, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide.
 17. A pharmaceutical composition comprising an agent identified by the method of claim 16 and a pharmaceutically acceptable carrier therefor.
 18. A method for treating a disease or condition mediated by a human enzyme protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim
 16. 19. A method for identifying a modulator of the expression of a peptide of claim 2, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide.
 20. An isolated human enzyme peptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence shown in SEQ ID NO:2.
 21. A peptide according to claim 20 that shares at least 90 percent homology with an amino acid sequence shown in SEQ ID NO:2.
 22. An isolated nucleic acid molecule encoding a human enzyme peptide, said nucleic acid molecule sharing at least 80 percent homology with a nucleic acid molecule shown in SEQ ID NOS: 1 or
 3. 23. A nucleic acid molecule according to claim 22 that shares at least 90 percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or
 3. 